On-slide staining by primer extension

ABSTRACT

A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.

CROSS-REFERENCING

This patent application claims the benefit of U.S. provisionalapplication Ser. No. 62/015,799, filed Jun. 23, 2014, and U.S.non-provisional application Ser. No. 14/560,921, filed on Dec. 4, 2014,which patent applications are incorporated by reference herein in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contractW81XWH-12-1-0591 awarded by the Department of Defense and undercontracts GM104148 and HHSN268201000034C awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND

Several major approaches have been used so far for single-cell antigencytometry. Among the most popular are single cell PCR, fluorescenceactivated flow cytometry, mass cytometry and single cell sequencing.These (fluorescence and mass-based cytometry) approaches are limitedfrom either inability to breach the multiplexing levels of more than 100parameters per analyte (cell in this case) or from inability to achievehigh throughput (single cell sequencing). Also these methods are notappropriate or readily modified to enable cell multiplexed analysis ofarchived tissues and slide based samples.

Disclosed herein are several related methods for capture agent detectionthat are based on labeling the capture agent with DNA and subsequentdetection of this DNA by primer extension.

SUMMARY

A method for analyzing a planar sample is provided. In certainembodiments, the method may comprise: (a) labeling the planar sample(e.g., a tissue section) with a capture agent (e.g., an antibody or anoligonucleotide probe) in a way that produces a labeled sample in which:(i) the capture agent is linked to a double-stranded nucleic acid thatcomprises a first strand and a second strand; and (ii) the 3′ end or 5′end of either the first strand or the second strand is extendible usingthe other strand as a template; (b) contacting the labeled sample withi. a polymerase and a nucleotide mix and/or ii. a labeledoligonucleotide and a ligase, thereby adding one or more nucleotidesand/or a labeled oligonucleotide to an one of the strands of thedouble-stranded nucleic acid; and (c) reading a fluorescent signalgenerated by addition of the one or more nucleotides and/oroligonucleotide to one of the strands of the double-stranded nucleicacid using fluorescence microscopy, thereby producing an image showingthe pattern of binding of the capture agent to the planar sample.

The method may be implemented in a variety of different ways. Forexample, in some embodiments, step (b) may contacting the labeled samplewith a polymerase and a nucleotide mix that comprises a fluorescentnucleotide, thereby adding the fluorescent nucleotide to one of thestrands (i.e., the top strand or the bottom strand, whichever strand hasthe extendible 3′ end) of the double-stranded nucleic acid; and step (c)may comprise reading a fluorescent signal generated by addition of thefluorescent nucleotide to one of the strands (i.e., the top strand orthe bottom strand, whichever strand has the extendible 3′ end) of thedouble-stranded nucleic acid. In this embodiment, the fluorescent signalmay: i. emitted directly from the added nucleotide; ii. a FRET signalgenerated by energy transfer between two fluorescent nucleotides thatare added to a 3′ end of one of the strands; or iii. a FRET signalgenerated by energy transfer between a first added fluorescentnucleotide (i.e., a fluorescent nucleotide that has been added to one ofthe strands) and a second fluorescent nucleotide that is already presentin one of the strands.

In alternative embodiments, step (b) comprises contacting the labeledsample with a ligase and a labeled oligonucleotide, thereby adding thelabeled oligonucleotide to the 3′ or 5′ end of one of the strands of thedouble-stranded nucleic acid; and step (c) comprises reading afluorescent signal generated by ligation of the labeled oligonucleotideto one of the strands of the double-stranded nucleic acid. In somecases, an extendible 3′ end may be extended by a polymerase, and ligatedto a labeled oligonucleotide. In these embodiments, the fluorescentsignal may be: i. emitted directly from the added nucleotide; ii. a FRETsignal generated by energy transfer between two fluorescent nucleotidesthat are added to one of the strands; or iii. a FRET signal generated byenergy transfer between a first fluorescent nucleotide added one of thestrands and a second fluorescent nucleotide that is already present inthe other strand.

In some embodiments, extension of one of the strands removes a quencherfrom a quenched fluorescently labeled oligonucleotide that is hybridizedto the other strand, downstream from the first strand.

In some embodiments, the first strand is a rolling circle amplification(RCA) product, and the second strand comprises oligonucleotides that arehybridized to multiple sites in the RCA product.

In other embodiments, the first strand is an oligonucleotide, and thesecond strand is a second oligonucleotide that is hybridized to thefirst oligonucleotide. In these embodiments, the oligonucleotides may bedesigned to produce a 5′ overhang such that the 3′ end of the firststrand oligonucleotide is extendible using the other oligonucleotide asa template. In other embodiments, the oligonucleotides may be designedto produce a 3′ overhang such that the 5′ end of the first strandoligonucleotide is extendible by ligation, using the otheroligonucleotide as a template

In any embodiment, the planar sample may be a tissue section, e.g., aformalin-fixed, paraffin-embedded (FFPE) tissue section.

Also provided herein is a capture agent that is linked to adouble-stranded nucleic acid, wherein: (i) the double-stranded nucleicacid comprises a first strand and a second strand; (ii) the captureagent is linked to the first strand; and (iii) the 3′ end or 5′ end ofeither the first strand or the second strand is extendible using theother strand as a template.

Also provided herein is a capture agent composition comprising aplurality of capture agents that recognize different complementarysites, wherein: each of the capture agents is linked to adouble-stranded nucleic acid that comprises a first strand and a secondstrand; the capture agents are linked to a double-stranded nucleic acidby the first strand; the 3′ end or 5′ end of the first or second strandis extendible using the other strand as a template; and the templatesimmediately downstream of the extendible ends are different for each ofthe capture agents. In these embodiments, the sequence of the firststrand is the same for each of the capture agents; and the sequence ofthe second strand is different for each of the capture agents.

In embodiments that use a reversible terminator (“reversible terminator”approach), the templates immediately adjacent to the template at theextendible 3′ end may be of the formula 3′-N_(4n)N₁/N₂/N₃-5′ optionallyfollowed by short stretch (e.g., 1-5 residues) of random nucleotides onthe 5′ end to increase the overall polymerase residence on the DNAduplex, where N₁, N₂, N₃ and N₄ are different nucleotides selected fromG, A, T and C and n is 0, 1 or more. In some cases, the populationcontains single nucleotide overhangs of nucleotides N₁, N₂ and N₃ or thepopulation of overhangs comprises two nucleotide overhangs of sequence3′-N₄N₁-5′, 3′-3′-N₄N₂-5′ and 3′-N₄N₃-5′-5′ and, optionally overhangs ofsequence, 3′-N₄N₄N₁-5′, 3′-N₄N₄N₂-5′ and 3′-N₄N₄N₃-5′ and so on (e.g.,four nucleotide overhangs of sequence 3′-N₄N₄N₄N₁-5′, 3′-N₄N₄N₄N₂-5′ and3′-N₄N₄N₄N₃-5′). A population of oligonucleotides or RCA products havingsequences that are defined by any of these formulas is also provided. InRCA embodiments, the sequence may be found in each repeat of an RCAproduct.

In these embodiments, the templates immediately adjacent to theextendible 3′ end may be of a more general formula 3′-XN₁/N₂/N₂-5′,where N₁, N₂, N₃ are different nucleotides selected from G, A, T and Cand X is a nucleotide stretch of bases Xi (such that Xi are differentnucleotides selected from G, A, T and C) of random composition andlength. In some cases, the population may comprise comprises twonucleotide overhangs of sequence 3′-X₁N₁-5′, 3′-X₁N₂-5′ and 3′-X₁N₃-5′and, optionally overhangs of sequence, 3′-N₁X₁X₂-5′, 3′-N₂X₁X₂-5′ and3′-N₃X₁X₂-5′ and so on (e.g., four nucleotide overhangs of sequence3′-N₁X₁X₂X₃-5′, 3′-N₂X₁X₂X₃-5′ and 3′-N₃X₁X₂X₃-5′). In many embodiments,this population additionally contains single nucleotide overhangs ofnucleotides N₁, N₂ and N₃. A population of oligonucleotides or RCAproducts having sequences that are defined by any of these formulas isalso provided. In RCA embodiments, the sequence may be found in eachrepeat of an RCA product.

In embodiments that rely on a “missing base” approach, the templateimmediately adjacent to the extendible 3′ end may be of the formula3′-YN₁/N₂-5′, optionally followed by short stretch (e.g., 1-5 residues)of random nucleotides on the 5′ end to increase the overall polymeraseresidence on the DNA duplex, wherein Y is a nucleotide sequence oflength n (n is 0, 1 or more) composed of bases N₃ and N₄, whereinnucleotide N₃ is in odd positions and nucleotide N₄ is in evenpositions, counting from the start of the overhang and N₁, N₂, N₃ and N₄are different nucleotides selected from G, A, T and C. For example, insome cases, the population may comprise 5′ overhangs of sequence3′-N₁-5′ and 3′-N₂-5′ or optionally 3′-N₃N₁-5′ and 3′-N₃N₂-5′ or3′-N₃N₄N₁-5′ and 3′-N₃N₄N₂-5′ and, optionally, overhangs of sequence3′-N₃N₄N₃N₁-5′ and 3′-N₃N₄N₃N₂-5′ and so on (e.g., overhangs of sequence3′-N₃N₄N₃N₄N₁-5′ and 3′-N₃N₄N₃N₄N₂-5′ and then 3′-N₃N₄N₃N₄N₃N₁-5′ and3′-N₃N₄N₃N₄N₃N₂-5′). A population of oligonucleotides or RCA productshaving sequences that are defined by any of these formulas is alsoprovided. In RCA embodiments, the sequence may be found in each repeatof an RCA product.

In these embodiments the template immediately adjacent to the extendible3′ end may also be of a more general formula 3′-YN₁/N₂-5′, wherein Y isa nucleotide sequence of length n (n is 0, 1 or more) composed ofalternating random length stretches of bases N₃ and N₄ such that theorder number of N₃—stretches is odd and of N₄ stretches is even andwherein N₁, N₂, N₃ and N₄ are different nucleotides selected from G, A,T and C. For example, the population may comprise overhangs of sequence3′-N₁-5′ and 3′-N₂-5′ or optionally 3′-N₃N₃N₁-5′ and 3′-N₃N₃N₂-5′ or3′-N₃N₃N₄N₁-5′ and 3′-N₃N₃N₄N₂-5′ and, optionally, overhangs of sequence3′-N₃N₃N₃N₃N₄N₄N₃N₃N₃N₁-5′ and 3′-N₃N₃N₃N₃N₄N₄N₃N₃N₃N₁-5′ and so on). Apopulation of oligonucleotides or RCA products having sequences that aredefined by any of these formulas is also provided. In RCA embodiments,the sequence may be found in each repeat of an RCA product.

A method for analyzing a tissue sample is also provided. In theseembodiments, the method may comprise (a) labeling a planar sample withthe above-described capture agent composition; (b) contacting thelabeled sample with i. a polymerase and either an incomplete nucleotidemix or a nucleotide mix that comprises a reversible terminatornucleotide and/or ii. a labeled oligonucleotide and a ligase; and (c)reading, using fluorescence microscopy, a fluorescent signal generatedby addition a nucleotide or a labeled oligonucleotide to some but notall of the capture agents.

In these embodiments, the method may comprises: (c) contacting theplanar sample with a polymerase and: (i) a nucleotide mix that comprisesfluorescent nucleotides that are complementary to N₁, N₂ and N₃ and areversible terminator nucleotide that is complementary to N₄ or (ii) anucleotide mix that comprises fluorescent nucleotides that arecomplementary to N₁, and N₂, an unlabeled nucleotide that iscomplementary to N₃, and no nucleotide that is complementary to N₄,thereby adding fluorescent nucleotides onto the double-stranded nucleicacids of some but not all of the capture agents; and (d) reading, usingfluorescence microscopy, a fluorescent signal generated by addition of afluorescent nucleotide to some but not all of the capture agents.

In some embodiments, the templates immediately adjacent to theextendible 3′ end are of the formula 3′-N_(4n)N₁/N₂/N₃, wherein N₁, N₂,N₃ and N₄ are different nucleotides selected from G, A, T and C and n is1 or more; and step (c) comprises contacting the planar sample with apolymerase and a nucleotide mix that comprises fluorescent nucleotidesthat are complementary to N₁, N₂ and N₃ and a reversible terminatornucleotide that is complementary to N₄.

In some embodiments, this method may further comprise: (e) inactivatingthe fluorescent signal, deprotecting the reversible terminatornucleotide and blocking the sample; and (f) repeating steps (c) and (d).In some cases, step (f) may comprise repeating steps (c), (d) and (e)multiple times.

In some embodiments, the templates immediately adjacent to theextendible 3′ end may be of the formula 3′-YN₁/N₂-5′, optionallyfollowed by short stretch (e.g., 1-5 nucleotides) of random nucleotideson the 5′ end to increase the overall polymerase residence on the DNAduplex, wherein Y is composed of alternating stretches of bases N₃ andN₄, and wherein N₁, N₂, N₃ and N₄ are different nucleotides selectedfrom G, A, T and C.

In these embodiments, the method may comprise (e) inactivating thefluorescent signal and contacting the planar sample with a polymeraseand a an unlabeled nucleotide that is complementary to N₄; and (f)repeating steps (c) and (d). In certain cases, step (f) may compriserepeating steps (c), (d) and (e) multiple times.

In alternative embodiments, the double-stranded oligonucleotides mayeach comprise a fluorescently labeled oligonucleotide hybridized to thesecond strand downstream from first strand, wherein the fluorescentlylabeled oligonucleotide comprises a quencher and extension of the firststrand removes the quencher from some but not all of the quenchedfluorescently labeled oligonucleotides, thereby generating a fluorescentsignal for some but not all of the capture agents.

In other embodiments, the capture agent is linked to a single strandedoligonucleotide, which can be either unlabeled or labeled with FRETacceptor fluorophore. Such a single stranded nucleotide incorporates adedicated sequence that hybridizes to a complementary oligonucleotidewhich is to be extended with unlabeled base or with a base labeled witha FRET excitation fluorophore, thereby generating a fluorescent signalfor some but not all of the capture agents.

In some embodiments, a method for analyzing a planar sample. In someembodiments, the method comprises: (a) labeling the planar sample with acapture agent to produce a labeled sample, wherein: (i) the captureagent is linked to a double-stranded nucleic acid that comprises a firststrand and a second strand; and (ii) a 3′ end or 5′ end of either thefirst strand or the second strand is extendible using the other strandas a template; (b) contacting the labeled sample with i. a polymeraseand a plurality of nucleotides and/or ii. a labeled oligonucleotide anda ligase, thereby adding one or more nucleotides of the plurality ofnucleotides and/or a labeled oligonucleotide to an end of one of thestrands of the double-stranded nucleic acid; and (c) reading a signalgenerated by addition of the one or more nucleotides and/or labeledoligonucleotide to one of the first strand or the second strand of thedouble-stranded nucleic acid. In some embodiments, the signal may be afluorescent signal. In some embodiments, the reading may comprisesfluorescence microscopy. Any embodiment, the method may further compriseproducing an image showing the pattern of binding of the capture agentto the planar sample.

In any embodiment, step (b) may comprise contacting the labeled samplewith a polymerase and a plurality of nucleotides that comprises afluorescent nucleotide, thereby adding the fluorescent nucleotide to oneof the first strand or the second strand of the double-stranded nucleicacid; and step (c) comprises reading a fluorescent signal generated byaddition of the fluorescent nucleotide to one of the first strand or thesecond strand of the double-stranded nucleic acid. In these embodiment,wherein the fluorescent signal may be: i. emitted directly from theadded nucleotide; ii. a FRET signal generated by energy transfer betweentwo fluorescent nucleotides of the plurality of fluorescent nucleotidesthat are added to one of the first strand or second strand of thedouble-stranded nucleic acid; or iii. a FRET signal generated by energytransfer between the added fluorescent nucleotide and a secondfluorescent nucleotide that is present in one of the first strand orsecond strand double-stranded nucleic acid.

In any embodiment, the method step (b) may comprise contacting thelabeled sample with a ligase and a labeled oligonucleotide, therebyadding the labeled oligonucleotide to one of the first strand or secondstrand of the double-stranded nucleic acid; and step (c) comprisesreading a fluorescent signal generated by addition of the labeledoligonucleotide to one of the first strand or second strand of thedouble-stranded nucleic acid. In this embodiment, the fluorescent signalmay be: i. emitted directly from the added labeled nucleotide; ii. aFRET signal generated by energy transfer between two labeled nucleotidesthat are added to one of the first strand or second strand of thedouble-stranded nucleic acid; or iii. a FRET signal generated by energytransfer between the labeled nucleotide added to one of the first strandand second strand of the double-stranded nucleic acid and a secondlabeled nucleotide that is present in the other strand. In theseembodiments, the labeled nucleotide may comprise a fluorescentnucleotide.

In any embodiment, extension of one of the first strand or second strandof the double-stranded nucleic acid may remove a quencher from aquenched fluorescently labeled oligonucleotide that is hybridized to theother strand, downstream from the first strand.

In any embodiment, the first strand of the double-stranded nucleic acidmay be a rolling circle amplification (RCA) product, and the secondstrand of the double-stranded nucleic acid comprises oligonucleotidesthat are hybridized to multiple sites in the RCA product.

In any embodiment, the first strand of the double-stranded nucleic acidmay be a first oligonucleotide, and the second strand of thedouble-stranded nucleic acid is a second oligonucleotide that ishybridized to the first oligonucleotide.

In any embodiment, the planar sample may be a formalin-fixed,paraffin-embedded (FFPE) section.

In any embodiment, the capture agent may be an antibody, an aptamer, oran oligonucleotide probe.

A capture agent that is linked to a double-stranded nucleic acid is alsoprovided. In some embodiments, (i) the double-stranded nucleic acidcomprises a first strand and a second strand; (ii) the capture agent islinked to the first strand; and (iii) the 5′ end or the 3′ end of eitherthe first strand or the second strand is extendible using the otherstrand as a template.

Also provided is a capture agent composition comprising a plurality ofcapture agents that each recognize different complementary sites. Inthese embodiments, each of the plurality of capture agents may be linkedto a double-stranded nucleic acid that comprises a first strand and asecond strand; the 5′ end or 3′ end of the first or second strand may beextendible using the other strand as a template; and the templatesimmediately downstream of the extendible ends may be different for eachof the plurality of capture agents. In these embodiments, the sequenceof the first strand may be the same for each of the plurality of captureagents; and the sequence of the second strand may be different for eachof the plurality of capture agents.

In some embodiments, the templates immediately adjacent to theextendible 3′ ends may be of the formula 3′-N_(4n)N₁/N₂/N₃, wherein N₁,N₂, N₃ and N₄ are different nucleotides selected from G, A, T and C andn is 1 or more.

In some embodiments, the templates immediately adjacent to theextendible 3′ ends may be of the formula 3′-YN₁/N₂-5′, optionallyfollowed by a short stretch of random nucleotides on the 5′ end toincrease the overall polymerase residence on the DNA duplex, wherein Yis composed of alternating stretches of N₃ and N₄, and wherein N₁, N₂,N₃ and N₄ are different nucleotides selected from G, A, T and C.

A method for analyzing a planar sample is provided. This method maycomprise (a) labeling the planar sample with a capture agent compositionsummarized above; (b) contacting the labeled sample with i. a polymeraseand either an incomplete nucleotide mix or a nucleotide mix thatcomprises a reversible terminator nucleotide, thereby adding anucleotide to the plurality of capture agents; and/or ii. a labeledoligonucleotide and a ligase, thereby adding a labeled oligonucleotideto the plurality of capture agents; and (c) reading a signal generatedby addition of the nucleotide or the labeled oligonucleotide to some butnot all of the plurality of capture agents. In these embodiments, thesignal may be a fluorescent signal. In some embodiments, the reading maybe done by fluorescent microscopy.

In some embodiments, the method may be done by (b) contacting the planarsample with a polymerase and: (i) a nucleotide mix that comprises aplurality of fluorescent nucleotides that are complementary to N₁, N₂and N₃ and a reversible terminator nucleotide that is complementary toN₄; or (ii) a nucleotide mix that comprises a plurality of fluorescentnucleotides that are complementary to N₁, and N₂, an unlabelednucleotide that is complementary to N₃, and no nucleotide that iscomplementary to N₄, thereby adding fluorescent nucleotides onto thedouble-stranded nucleic acids of some but not all of the plurality ofcapture agents; and (c) reading, using fluorescence microscopy, afluorescent signal generated by addition of the fluorescent nucleotidesto the double-stranded nucleic acids of some but not all of theplurality of capture agents. In these embodiments, the templatesimmediately adjacent to the extendible 3′ end may be of the formula3′-N_(4n)N₁/N₂/N₃, wherein N₁, N₂, N₃ and N₄ are different nucleotidesselected from G, A, T and C and n is 1 or more; and step (b) comprisescontacting the planar sample with a polymerase and a nucleotide mix thatcomprises a plurality of fluorescent nucleotides that are complementaryto N₁, N₂ and N₃ and a reversible terminator nucleotide that iscomplementary to N₄. In these embodiments, the method may furthercomprise: (d) inactivating the fluorescent signal, (e) optionally,deprotecting the reversible terminator nucleotide; (f) blocking thesample; and (g) repeating steps (b) and (c). In some embodiment, step(g) may comprise repeating steps (b)-(f) multiple times.

In some embodiments, the templates immediately adjacent to theextendible 3′ end may be of the formula 3′-YN₁/N₂-5′, optionallyfollowed by a short stretch of random nucleotides on the 5′ end toincrease the overall polymerase residence on the DNA duplex, wherein Yis composed of alternating stretches of N₃ and N₄, and wherein N₁, N₂,N₃ and N₄ are different nucleotides selected from G, A, T and C. Inthese embodiments, the method may further comprise: (d) inactivating thefluorescent signal; (e) contacting the planar sample with a polymeraseand an unlabeled nucleotide that is complementary to N₄; and (f)repeating steps (b) and (c). In some cases, step (f) may compriserepeating steps (b)-(e) multiple times.

In some embodiments, the double-stranded nucleic acids each comprise afluorescently labeled oligonucleotide hybridized to the second stranddownstream from the first strand, wherein the fluorescently labeledoligonucleotide comprises a quencher and extension of the first strandremoves the quencher from some but not all of the quenched fluorescentlylabeled oligonucleotides, thereby generating a fluorescent signal forsome but not all of the plurality of capture agents.

In some embodiments, extension of the double-stranded nucleic acidcomprises contacting the planar sample with a mixture of labeled andunlabeled oligonucleotides and a ligase.

In any embodiment, the plurality of capture agents may be selected fromthe group consisting of: antibodies, aptamers, and oligonucleotideprobes.

A kit is also provided. In these embodiments, the kit may comprise: (a)one or more capture agents, wherein the one or more capture agents canspecifically bind to complementary sites in a planar sample. (b) one ormore double-stranded nucleic acids comprising a first strand a secondstrand, wherein each of the one or more capture agents is linked to thedouble-stranded nucleic acid, and wherein a 5′ end or 3′ end of eitherthe first strand or the second strand is extendible using the otherstrand as a template. In some embodiments, the kit may further comprisea polymerase or ligase. In some embodiments, the kit may furthercomprise a nucleotide mix comprising at least one of a fluorescentnucleotide, an unlabeled nucleotide, and a reversible terminatornucleotide. In some embodiments, the one or more capture agents may beselected from the group consisting of: an antibody, an aptamer and anoligonucleotide probe.

In some aspects, a method is provided for analyzing a planar sample. Insome cases, the method comprises incubating the planar sample with acapture agent under conditions by which the capture agent specificallybinds to complementary sites in the planar sample. In some cases, thecapture agent is linked to a double-stranded oligonucleotide thatcomprises a first strand and a second strand. In some cases, a 3′ end ofthe first strand is recessed relative to a 5′ end of the second strand,thereby producing an overhang. In some cases, the method comprisescontacting the planar sample with a polymerase and a plurality ofnucleotides, thereby adding one or more nucleotides of the plurality ofnucleotides to the overhang. In some cases, the method comprises readinga signal generated by addition of the one or more nucleotides to theoverhang. In some cases, the plurality of nucleotides comprises aplurality of fluorescent nucleotides. In some cases, a fluorescentnucleotide of the plurality of nucleotides is added to the overhang. Insome cases, the signal comprises a fluorescent signal. In some cases,the fluorescent signal is emitted directly from the fluorescentnucleotide added to the overhang. In other cases, two of the pluralityof fluorescent nucleotides are added to the overhang. In this example,the fluorescent signal is a FRET signal generated by energy transferbetween the two of the plurality of fluorescent nucleotides added to theoverhang. In an alternative example, the fluorescent signal is a FRETsignal generated by energy transfer between the fluorescent nucleotidefrom the plurality of fluorescent nucleotides added to the overhang anda fluorescent nucleotide that is present in the second strand. In somecases, extension of the first strand removes a quencher from a quenchedfluorescently labeled oligonucleotide that is hybridized to the secondstrand, downstream from the first strand. In some cases, the planarsample is a formalin-fixed, paraffin-embedded (FFPE) section. In somecases, the capture agent is linked to the double-strandedoligonucleotide by a 5′ end of the first strand. In other cases, thecapture agent is linked to the double-stranded oligonucleotide by a 3′end of the second strand. In some cases, the method further comprisescrosslinking the capture agent to the planar sample. In some cases, thereading comprises fluorescence microscopy. In some cases, the methodfurther comprises producing an image showing a pattern of binding of thecapture agent to the planar sample. In some cases, the one or morenucleotides of the plurality of nucleotides is added to the overhang byprimer extension. In some cases, the capture agent is an antibody, anaptamer or an oligonucleotide probe.

In some aspects, a composition is provided comprising a plurality ofcapture agents that specifically bind to different complementary sitesin a planar sample. In some cases, each of the plurality of captureagents is linked to a double-stranded oligonucleotide that comprises afirst strand and a second strand. In some cases, a 3′ end of the firststrand in each of the double-stranded oligonucleotides is recessedrelative to a 5′ end of the second strand, thereby producing anoverhang. In some cases, the overhang is different for each of theplurality of capture agents. In some cases, each of the plurality ofcapture agents is linked to the double-stranded oligonucleotide by a 5′end of the first strand. In other cases, each of the plurality ofcapture agents is linked to the double-stranded oligonucleotide by a 3′end of the second strand. In some cases, a sequence of the first strandis the same for each of the plurality of capture agents and a sequenceof the second strand is different for each of the plurality of captureagents. In some cases, the overhang is of the formula 3′-N4nN1/N2/N3,wherein N1, N2, N3 and N4 are different nucleotides selected from G, A,T and C and n is 1 or more. In other cases, the overhang is of theformula 3′-YN1/N2-5′, optionally followed by a short stretch of randomnucleotides on the 5′ end of the first strand to increase the overallpolymerase residence on the DNA duplex, wherein Y is composed ofalternating stretches of N3 and N4, and wherein N1, N2, N3 and N4 aredifferent nucleotides selected from G, A, T and C. In some cases, Y is anucleotide sequence of length n and wherein n is 0, 1, or more. In somecases, the order number of N3 stretches is odd and wherein the ordernumber of N4 stretches is even. In some cases, the planar sample is aformalin-fixed, paraffin-embedded section (FFPE). In some cases, theplurality of capture agents are antibodies, aptamers, or oligonucleotideprobes.

In some aspects, a method is provided for analyzing a planar sample. Insome cases, the method comprises incubating the planar sample with thecomposition described above under conditions by which each of theplurality of capture agents specifically bind to different complementarysites in the planar sample. In some cases, the method comprisescontacting the planar sample with a polymerase and a plurality ofnucleotides, thereby adding one or more nucleotides of the plurality ofnucleotides to the overhang of some, but not all, of the plurality ofcapture agents. In some cases, the method comprises reading a signalgenerated by addition of the one or more nucleotides from the pluralityof nucleotides to the overhang of some, but not all, of the plurality ofcapture agents. In some cases, the method further comprises crosslinkingthe plurality of capture agents to the planar sample.

In some cases, the plurality of nucleotides comprises an incompletenucleotide mix or a nucleotide mix comprising a reversible terminatornucleotide. In some cases, the signal comprises a fluorescent signal. Insome cases, the reading comprises fluorescence microscopy. In somecases, the method further comprises producing an image showing a patternof binding of the plurality of capture agents to the planar sample. Insome cases, the plurality of nucleotides comprises: (i) a plurality offluorescent nucleotides that are complementary to N1, N2 and N3, and areversible terminator nucleotide that is complementary to N4; or (ii) aplurality of fluorescent nucleotides that are complementary to N1 andN2, an unlabeled nucleotide that is complementary to N3, and nonucleotide that is complementary to N4. In some cases, a fluorescentnucleotide of the plurality of fluorescent nucleotides is added to theoverhang of some, but not all, of the plurality of capture agents. Insome cases, the signal comprises a fluorescent signal generated byaddition of the fluorescent nucleotide of the plurality of fluorescentnucleotides to some, but not all, of the plurality of capture agents. Insome cases, the reading comprises fluorescence microscopy. In somecases, the method further comprises producing an image showing thepattern of binding of the plurality of capture agents to the planarsample. In some cases, the overhangs are of the formula 3′-N4nN1/N2/N3,wherein N1, N2, N3 and N4 are different nucleotides selected from G, A,T and C and n is 1 or more, and wherein the plurality of nucleotidescomprises a plurality of fluorescent nucleotides that are complementaryto N1, N2, N3 and a reversible terminator nucleotide that iscomplementary to N4. In some cases, the method further comprisesinactivating the fluorescent signal, optionally, deprotecting thereversible terminator nucleotide; blocking the planar sample; andrepeating the steps of contacting and reading. In some cases, therepeating further comprises repeating the steps of contacting, reading,inactivating, optionally deprotecting, and blocking a plurality oftimes. In other cases, the overhangs are of the formula 3′-YN1/N2-5′,optionally followed by a short stretch of random nucleotides on the 5′end of the first strand to increase the overall polymerase residence onthe DNA duplex, wherein Y is composed of alternating stretches of N3 andN4, and wherein N1, N2, N3 and N4 are different nucleotides selectedfrom G, A, T and C. In some cases, Y is a nucleotide sequence of lengthn and wherein n is 0, 1, or more. In some cases, the order number of N3stretches is odd and wherein the order number of N4 stretches is even.In some cases, the method further comprises inactivating the fluorescentsignal, contacting the planar sample with a polymerase and an unlabelednucleotide that is complementary to N4; and repeating the steps ofcontacting and reading. In some cases, the repeating comprises repeatingthe steps of contacting, reading, inactivating, and contacting aplurality of times. In some cases, each of the double-strandedoligonucleotides comprise a fluorescently labeled oligonucleotidehybridized to the second strand downstream from the first strand,wherein the fluorescently labeled oligonucleotide comprises a quencherand extension of the first strand removes the quencher from some, butnot all, of the quenched fluorescently-labeled oligonucleotides, therebygenerating a fluorescent signal for some, but not all, of the captureagents.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A-1B (A) schematically illustrates a detection reagent composed ofa combination of a capture agent that is conjugated to a double-strandedoligonucleotide. Upon detection and removal of unbound detection reagentthe binding pattern is rendered by polymerase driven primer extension.Panel (B) schematically illustrates three approaches for linking thecapture agent (an antibody in this case, but not excluding otherpossible capture agents) to a double stranded oligonucleotide (i.e., bychemical conjugation of the upper strand oligonucleotide to the captureagent; using streptavidin as an intermediate to connect biotinylatedantibody and biotinylated oligonucleotide; and by linking biotinylatedoligonucleotide to antibody chemically conjugated to streptavidin).

FIG. 2 schematically illustrates examples of capture agents that arebound to double-stranded oligonucleotides that have different overhangs.Such different overhangs represent a strategy to increase signalharvested from a particular capture agent by multiplication of positionsin lower strand oligonucleotide complementary to detector base (dU inthis case). The lower panel also shows how a different base labeled witha different fluorophore can be used as a FRET excitation pair for the“Detector” base. SEQ ID NOS: 1-4.

FIG. 3 schematically illustrates several cycles of a multiplexeddetection method that relies on reversible dye terminators.

FIG. 4 schematically illustrates several cycles of a multiplexeddetection method that relies on leaving out one of the four nucleotidesper cycle.

FIG. 5A-5D schematically illustrates an exemplary design ofoligonucleotide duplexes for “reversible terminator” and “missing base”multiplexing methods. SEQ ID NOS: 5-12.

FIG. 6 schematically illustrates an exemplary design of oligonucleotideduplexes for a strategy that allows one to reduce the length of thelower strand oligonucleotide, creating an overhang in the case of highlymultiplexed capture agent panels. SEQ ID NOS: 13-30.

FIG. 7 schematically illustrates an example of a detection method thatrelies on removing a quencher from a labeled oligonucleotide by nicktranslation. SEQ ID NOS: 31-35.

FIG. 8 schematically illustrates a multiplexed detection method thatrelies on removing quenchers from labeled oligonucleotides. Step 1: SEQID NOS 36-44, Step 2: SEQ ID NOS: 45-52, Step 3: SEQ ID NOS: 53-60, Step4: SEQ ID NOS: 61-67.

FIGS. 9A and 9B schematically illustrate an embodiment that relies oncyclical re-annealing of polymerase priming nucleotides and a variant ofthe same approach that utilizes FRET. SEQ ID NOS: 68-80.

FIG. 10 schematically illustrates an embodiment that relies on cyclicalre-annealing of polymerase priming nucleotides and a variant of the sameapproach that utilizes FRET. SEQ ID NOS: 81-86.

FIGS. 11A-11C shows an anti-CD4 antibody linked to oligonucleotideduplex designed for rendering staining by primer extension (panel A) anddata obtained from labeled population of spleen cells in suspension inthe absence of polymerase (panel B) and in the presence of polymerase(panel C). SEQ ID NOS: 87 and 88.

FIGS. 12A-12D shows data obtained from labeling by primer extension apopulation of spleen cells preattached on the slide. Cells wereco-stained with “regular” TCRb-FITC antibody and CD4 antibody linked tooligonucleotide duplex designed for rendering staining by primerextension.

FIGS. 13A-13D show schematic illustration of two capture agents CD4 andCD8 linked to oligonucleotide duplexes (panel A) and data obtained froma multiplexed method whereby staining by this capture agents wassequentially detected on spleen cells smeared on a slide using a“reversible terminator” method (panels C-D). SEQ ID NOS: 89-92.

FIG. 14 shows a schematic diagram of an experiment testing multiplexedstaining by “missing base” approach. Mouse spleen samples were barcodedby pan-leukocytic CD45 antibody conjugated to per sample specificoligonucleotide duplexes. Samples were mixed after staining and mixturewas resolved by sequential rendering of CD45-oligonucleotide variants.

FIG. 15 is 12 panels of images showing the first 6 cycles of renderingthe 30 populations barcoded by CD45 (as per scheme on FIG. 14). Twopopulations were co-detected per cycle of rendering. In each cyclecontrol image was acquired after fluorescence inactivation.

FIG. 16 illustrates enhanced antibody signal with rolling circleamplification. A. Antibody-DNA conjugate that consists of an antibody, acovalently linked linear linker oligonucleotide and a 5′-phosphorylatedpadlock nucleotide is used to stain the cellular antigens. Padlock probecontains the detection primer sequence (orange) followed by thefluorescent nucleotide incorporation site (T). B. Padlockoligonucleotide is treated with T4 DNA ligase, inducing itscircularization. C. Rolling circle amplification with strand-displacingphi29 DNA polymerase created repeats of the reverse-complement of thedetection primer sites (green). F-G. Staining of Mouse Spleen cells withantibody-DNA conjugate visualized by primer extension with dUTP-Cy5without the rolling circle amplification (F) and after rolling circleamplification (G).

FIG. 17 shows fluorescent images of cells, showing the staining of 22different antigens rendered by the iterative primer extension protocol.At each cycle one antigen-antibody-DNA complex incorporates dUTP—SS-Cy5fluorophore (red) and one complex incorporates dCTP—SS-Cy3 (green), allother complexes receive an unlabelled ‘walking’ base (dGTP on oddcycles, dATP on even cycles).

FIG. 18 shows A: multipanel design whereby antibody-DNA conjugates areincapable of polymerase extension because of 3′-dideoxy-terminatorbases, but each panel can be activated for extension independently ofothers by an addition of a panel-specific primer. B: 18 aliquotes ofmouse spleen cells were independently stained with different CD45antibody conjugates that were designed such. Aliquots 1-3 (panel 1) canbe detected by regular ABseq primer extension (top row), aliquots 4-6(panel 2) were be extended after addition of Spacer1 oligonucleotideprimer and aliquotes 7-9 (panel 3) can be extended after addition ofSpacer2 oligonucleotide primer. C: Results of image quantification.Intensities of individual cell intensities displayed as a barcodes, onecell for each row, red color representing higher staining intensity.Columns represent intensities of cells on each extension cycle. Thediagonal pattern shows the high specificity of spacer-based extensionand the absence of signal cross-talk between panels and extensioncycles.

FIG. 19 shows A: A pair of coincidence detection probes is hybridized tothe target RNA. Upstream oligonucleotide probe (Splint-primer) serves asa splint for circularization and ligation of the downstreamoligonucleotide probe (padlock). Padlock probe contains a detectionprimer sequence (lilac) followed by the fluorescent nucleotideincorporation site (red) B. Rolling circle amplification is initiated atthe 3′ end of the upstream probe and creates multiple copies of thereverse-complement of detection primer sequence (lilac). C. Detectionprimer is annealed to the multiple sites of the amplification product.D. Polymerase reaction with dUTP-Cy5 results incorporations. E-F: smalland bright puncta in NALM cells correspond to single HLADRA RNAmolecules, which are absent in the negative control Jurkat cells. Largered blobs present in both panels correspond to apoptotic cells thatnonspecifically bind the fluorescent nucleotide.

FIG. 20 shows an alternative method that relies on primer extension andthe ligation of a short, labeled oligonucleotide. Left side, from top tobottom: SEQ ID NOS: 93-108; right side, from top to bottom: SEQ ID NOS:109-124.

FIG. 21 depicts a system to enable a user to detect, analyze, andprocess images of samples.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

As used herein, the term “biological feature of interest” refers to anypart of a cell that can be indicated by binding to a capture agent.Exemplary biological features of interest include cell walls, nuclei,cytoplasm, membrane, keratin, muscle fibers, collagen, bone, proteins,nucleic acid (e.g., mRNA or genomic DNA, etc). fat, etc. A biologicalfeature of interest can also be indicated by immunohistological methods,e.g., a capture agent that is linked to an oligonucleotide. In theseembodiments, the capture agent binds to an site, e.g., a proteinepitope, in the sample. Exemplary epitopes include, but are not limitedto carcinoembryonic antigen (for identification of adenocarcinomas,cytokeratins (for identification of carcinomas but may also be expressedin some sarcomas) CD15 and CD30 (for Hodgkin's disease), alphafetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD117(for gastrointestinal stromal tumors), CD10 (for renal cell carcinomaand acute lymphoblastic leukemia), prostate specific antigen (forprostate cancer), estrogens and progesterone (for tumouridentification), CD20 (for identification of B-cell lymphomas), CD3 (foridentification of T-cell lymphomas). Complementary nucleic acidmolecules (e.g., DNA and/or RNA) in the sample provide bindingcomplementary sites for oligonucleotide probes.

As used herein, the term “multiplexing” refers to using more than onelabel for the simultaneous or sequential detection and measurement ofbiologically active material.

As used herein, the terms “antibody” and “immunoglobulin” are usedinterchangeably herein and are well understood by those in the field.Those terms refer to a protein consisting of one or more polypeptidesthat specifically binds an antigen. One form of antibody constitutes thebasic structural unit of an antibody. This form is a tetramer andconsists of two identical pairs of antibody chains, each pair having onelight and one heavy chain. In each pair, the light and heavy chainvariable regions are together responsible for binding to an antigen, andthe constant regions are responsible for the antibody effectorfunctions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,minibodies, single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Alsoencompassed by the term are Fab′, Fv, F(ab′)₂, and or other antibodyfragments that retain specific binding to antigen, and monoclonalantibodies. Antibodies may exist in a variety of other forms including,for example, Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e.bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)) and in single chains (e. g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.,Science, 242, 423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).

The term “specific binding” refers to the ability of a binding reagentto preferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between a binding reagent andanalyte when they are specifically bound in a capture agent/analytecomplex is characterized by a K_(D) (dissociation constant) of less than10⁻⁶M, less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than10⁻⁹ M, less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 2, at least 5, at least 10, at least 100, at least1000, at least 10,000, at least 100,000, at least 10⁶, at least 10⁷, atleast 10⁸ or at least 10⁹ or more members.

As used herein, the term “labeling” refers to attaching a detectablefluorophore to specific sites in a sample (e.g., sites containing anepitope for the antibody being used, for example) such that the presenceand/or abundance of the sites can be determined by evaluating thepresence and/or abundance of the label.

The term “labelling” refers to a method for producing a labeled samplein which any necessary steps are performed in any convenient order, aslong as the required labeled sample is produced. For example, in someembodiments and as will be exemplified below, the capture agent may bealready linked to a double-stranded nucleic acid prior to binding of theantibody to the sample, in which case a sample can be labeled usingrelatively few steps. In other embodiments, the capture agent may belinked to the first strand of the double stranded nucleic acid at thetime at which it is incubated with the sample. In these embodiments, thesecond strand of the double stranded nucleic acid may be hybridized tothe first strand of the double stranded nucleic acid after the antibodyhas bound to the sample. Along similar lines, the capture agent may belinked to a rolling circle amplification (RCA) primer at the time atwhich it is incubated with the sample. In these embodiments, thedouble-stranded nucleic acid may be produced by: a) hybridizing thesample with a padlock probe having ends that are complementary to theRCA primer, ligating the ends of the padlock probes together, andcopying the padlock probe by rolling circle amplification and b)hybridizing an oligonucleotide to the RCA product, as illustrated inFIG. 16. In this example, the RCA product is the first strand of thedouble-stranded nucleic acid, and the oligonucleotides that arehybridized to the RCA product are the second strand of thedouble-stranded nucleic acid. In many embodiments, the labeling step maycomprise crosslinking the capture agent to the planar sample so thatsubsequence manipulations can be done without the capture agentdisassociating from its complementary sites in the planar sample. Inthese embodiments, if the capture agent is linked to the double-strandednucleic acid prior to binding of the antibody to the sample, then thecrosslinking step may be done immediately after binding of the antibodyto the sample. In embodiments in which the capture agent is only linkedto the first strand (or an RCA primer for making the same) at the timeat which it is incubated with the sample, the sample may be cross-linkedafter binding of the antibody to the sample, and the double-stranded maybe produced after crosslinking.

As used herein, the term “planar sample” refers to a substantiallyplanar, i.e., two dimensional, material (e.g. glass, metal, ceramics,organic polymer surface or gel) that contains cells or any combinationsof biomolecules derived from cells, such as proteins, nucleic acids,lipids, oligo/polysachharides, biomolecule complexes, cellular organels,cellular debris or excretions (exosomes, microvesicles). A planarcellular sample can be made by, e.g., growing cells on a planar surface,depositing cells on a planar surface, e.g., by centrifugation, bycutting a three dimensional object that contains cells into sections andmounting the sections onto a planar surface, i.e., producing a tissuesection, absorbing the cellular components onto the surface that isfunctionalized with affinity agents (e.g. antibodies, haptens, nucleicacid probes), introducing the biomolecules into a polymer gel ortransferring them onto a polymer surface electrophoretically or by othermeans. The cells or biomolecules may be fixed using any number ofreagents including formalin, methanol, paraformaldehyde, methanol:aceticacid, glutaraldehyde, bifunctional crosslinkers such asbis(succinimidyl)suberate, bis(succinimidyl)polyethyleneglycole etc.This definition is intended to cover cellular samples (e.g., tissuesections, etc), electrophoresis gels and blots thereof, Western blots,dot-blots, ELISAs, antibody microarrays, nucleic acid microarrays etc.

As used herein, the term “tissue section” refers to a piece of tissuethat has been obtained from a subject, fixed, sectioned, and mounted ona planar surface, e.g., a microscope slide.

As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissuesection” refers to a piece of tissue, e.g., a biopsy that has beenobtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehydein phosphate buffered saline) or Bouin solution, embedded in wax, cutinto thin sections, and then mounted on a microscope slide.

As used herein, the term “spatially-addressable measurements” refers toa set of values that are each associated with a specific position on asurface. Spatially-addressable measurements can be mapped to a positionin a sample and can be used to reconstruct an image of the sample.

A “diagnostic marker” is a specific biochemical in the body which has aparticular molecular feature that makes it useful for detecting adisease, measuring the progress of disease or the effects of treatment,or for measuring a process of interest.

A “pathoindicative” cell is a cell which, when present in a tissue,indicates that the animal in which the tissue is located (or from whichthe tissue was obtained) is afflicted with a disease or disorder. By wayof example, the presence of one or more breast cells in a lung tissue ofan animal is an indication that the animal is afflicted with metastaticbreast cancer.

The term “complementary site” is used to refer to an epitope for anantibody or aptamer, or a nucleic acid molecule if the capture agent isan oligonucleotide probe. Specifically, if the capture agent is anantibody, then the complementary site for the capture agent is theepitope in the sample to which the antibody binds. If the capture agentis an oligonucleotide probe, then the complementary site for the captureagent is a complementary sequence in a DNA or RNA molecule in thesample.

The term “epitope” as used herein is defined as small chemical groups onthe antigen molecule that is bound to by an antibody. An antigen canhave one or more epitopes. In many cases, an epitope is roughly fiveamino acids or sugars in size. One skilled in the art understands thatgenerally the overall three-dimensional structure or the specific linearsequence of the molecule can be the main criterion of antigenicspecificity.

A “subject” of diagnosis or treatment is a plant or animal, including ahuman Non-human animals subject to diagnosis or treatment include, forexample, livestock and pets.

As used herein, the term “incubating” refers to maintaining a planarsample and capture agent under conditions (which conditions include aperiod of time, a temperature, an appropriate binding buffer and a wash)that are suitable for specific binding of the capture agent to molecules(e.g., epitopes or complementary nucleic acid) in the planar sample.

As used herein, the term “capture agent” refers to an agent that canspecifically bind to complementary sites in a planar sample. Exemplarycapture agents include, e.g., an antibody, an aptamer, and a nucleicacid (e.g., oligonucleotide) probe (which may be DNA or RNA) thathybridizes to a binding site. If antibodies are used, in many cases theantibodies may bind to protein epitopes. If nucleic acid probes areused, the nucleic acid probes may bind to, for example, genomic DNA orRNA (such that the location and abundance of intracellular RNAs can bedetected).

As used herein, the term “extendible”, in the context of, for example, a3′ end that is “extendible using the other strand as a template”, meansthat a polymerase or ligase can add to the 3′ end of a nucleic acidmolecule, where the template sequence that is immediately downstream ofthe 3′ end (i.e., on the other strand) determines which nucleotides (ifa polymerase is used) or oligonucleotide (if a ligase is used) is added.A “5′ end that is extendible using the other strand as a template” meansthat a ligase can add an oligonucleotide to the 5′ end of a nucleic acidmolecule, where the template sequence that is immediately downstream ofthe 5′ end (i.e., on the other strand) determines which oligonucleotideis added.

As used herein, the term “template sequence that is immediatelydownstream to the 3′ end” refers to the sequence on the other strandthat use used as a template for extending the 3′ end, starting with thefirst nucleotide. In embodiments in which the first strand is an RCAproduct, the template sequence that is immediately downstream of the 3′end may be a sequence in the RCA product. In embodiments in which thefirst strand is an oligonucleotide, the template sequence that isimmediately downstream of the 3′ end may be a 5′ overhang.

As used herein, the term “capture agent that is linked to a doublestranded nucleic acid” refers to a capture agent, e.g., an antibody oran oligonucleotide probe, that is non-covalently (e.g., via astreptavidin/biotin interaction) or covalently (e.g., via a clickreaction or the like) linked to an double-stranded nucleic acid (whichmay be composed of two single-stranded oligonucleotide strands that arehybridized together, or an RCA product that is hybridized to a pluralityof oligonucleotides) in a way that the capture agent can still bind toits binding site and the 3′ end of one of the nucleic acids isaccessible to a polymerase and/or ligase. The nucleic acid and thecapture agent may be linked via a number of different methods, includingthose that use maleimide or halogen-containing group, which arecysteine-reactive. The capture agent and the nucleic acid may be linkedat, proximal to or at the 5′ end of one of the strands of the doublestranded nucleic acid, proximal to or at the 3′ end of one of thestrands of the double stranded nucleic acid, or anywhere in-between.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g.,deoxyribonucleotides, ribonucleotides or a combination thereof, and maybe produced enzymatically or synthetically (e.g., PNA as described inU.S. Pat. No. 5,948,902 and the references cited therein) and which canhybridize with naturally occurring nucleic acids in a sequence specificmanner analogous to that of two naturally occurring nucleic acids, e.g.,can participate in Watson-Crick base pairing interactions.Naturally-occurring nucleotides include guanine, cytosine, adenine,thymine, uracil (G, C, A, T and U respectively). DNA and RNA have adeoxyribose and ribose sugar backbone, respectively, whereas PNA'sbackbone is composed of repeating N-(2-aminoethyl)-glycine units linkedby peptide bonds. In PNA various purine and pyrimidine bases are linkedto the backbone by methylene carbonyl bonds. A locked nucleic acid(LNA), often referred to as an inaccessible RNA, is a modified RNAnucleotide. The ribose moiety of an LNA nucleotide is modified with anextra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks”the ribose in the 3′-endo (North) conformation, which is often found inthe A-form duplexes. LNA nucleotides can be mixed with DNA or RNAresidues in the oligonucleotide whenever desired. The term “unstructurednucleic acid”, or “UNA”, is a nucleic acid containing non-naturalnucleotides that bind to each other with reduced stability. For example,an unstructured nucleic acid may contain a G′ residue and a C′ residue,where these residues correspond to non-naturally occurring forms, i.e.,analogs, of G and C that base pair with each other with reducedstability, but retain an ability to base pair with naturally occurring Cand G residues, respectively. Unstructured nucleic acid is described inUS20050233340, which is incorporated by reference herein for disclosureof UNA.

As used herein, the term “oligonucleotide” refers to a multimer of atleast 10, e.g., at least 15 or at least 30 nucleotides. In someembodiments, an oligonucleotide may be in the range of 15-200nucleotides in length, or more.

As used herein, the term “reading” in the context of reading afluorescent signal, refers to obtaining an image by scanning or bymicroscopy, where the image shows the pattern of fluorescence as well asthe intensity of fluorescence in a field of view.

As used herein, the term “primer” is an oligonucleotide, either naturalor synthetic, that is capable, upon forming a duplex with apolynucleotide template, of acting as a point of initiation of nucleicacid synthesis and being extended from its 3′ end along the template sothat an extended duplex is formed. The sequence of nucleotides addedduring the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. A primer may be at least 10, e.g., at least 15 or at least30 nucleotides in length.

As used herein, the term “single nucleotide 5′ overhang” refers to a 5′overhang, where the overhang is a single nucleotide in length. Likewise,a “two nucleotide 5′ overhang” is a 5′ overhang, where the overhang istwo nucleotides in length. The 3′ end is recessed in a 5′ overhang.

In certain cases, the various nucleotides of an overhang may be referredto by their position, e.g., “first position” and “second position”. Inthese cases, the “position” is relative to the recessed 3′ end. As such,in a multiple base 5′ overhang, the “first” position of the overhang isimmediately adjacent to the recessed 3′ end and the “second” position ofthe overhang is immediately adjacent to the first position.

In certain cases, the complementary strands of a double strandedoligonucleotide or nucleic acid may be referred to herein as being the“first” and “second” or the “top” and “bottom” strands. The assignmentof a strand as being a “top” or “bottom” strand is arbitrary and doesnot imply any particular orientation, function or structure.

As used herein, the term “signal generated by”, in the context ofreading a fluorescent signal generated by addition of the fluorescentnucleotide, refers to a signal that is emitted directly from thefluorescent nucleotide, a signal that is emitted indirectly via energytransfer to another fluorescent nucleotide (i.e., by FRET).

As used herein, the term “fluorescently labeled oligonucleotidecomprising a quencher” refers to an oligonucleotide that contains afluorophore and a quencher, wherein the quencher quenches thefluorophore in the same oligonucleotide.

As used herein, the term “different” in the context of different 5′overhangs that are different, refers to overhangs that have a differentsequence. Overhangs of different lengths (e.g., GATC vs GAT) implicitlyhave a different sequence, even through one sequence may be encompassedby the other.

As used herein, the term “overhang” refers to a structure in which onestrand of a double stranded nucleic acid ends such that nucleic acidsynthesis can be initiated from that strand by a polymerase (or anoligonucleotide can be ligated to the end by a ligase) using the otherstrand as a template.

As used herein, the term “adding to the extendible 3′ end”, in thecontext of adding one or more nucleotides or an oligonucleotide to anextendible 3′ end, refers to adding nucleotides (or an oligonucleotide)to an extendible 3′ end using the other strand as a template (e.g.,adding to the recessed 3′ end of a 5′ overhang using the overhang as atemplate).

As used herein, the term “template of the formula 3′-N_(4n)N₁/N₂/N₃-5′followed by an optional short stretch (e.g., 1-5 residues) of randomnucleotides on the 5′ end to increase the overall polymerase residenceon the DNA duplex, where N₁, N₂, N₃ and N₄ are different nucleotidesselected from G, A, T and C and n is 0, 1 or more”, refers to apopulation of sequences that potentially contains single nucleotideoverhangs of nucleotides N₁, N₂ and N₃ or the population of overhangscomprises two nucleotide overhangs of sequence 3′-N₄N₁-5′, 3′-N₄N₂-5′and 3′-N₄N₃-5′-5′ and, optionally overhangs of sequence, 3′-N₄N₄N₁-5′,3′-N₄N₄N₂-5′ and 3′-N₄N₄N₃-5′ and so on (e.g., four nucleotide overhangsof sequence 3′-N₄N₄N₄N₁-5′, 3′-N₄N₄N₄N₂-5′ and 3′-N₄N₄N₄N₃-5′).

As used herein, the term “template of the formula 3′-YN₁/N₂-5′,optionally followed by short stretch (e.g., 1-5 residues) of randomnucleotides on the 5′ end to increase the overall polymerase residenceon the DNA duplex, wherein Y is a nucleotide sequence of length n (n is0, 1 or more) composed of bases N₃ and N₄, wherein nucleotide N₃ is inodd positions and nucleotide N₄ is in even positions, counting from thestart of the overhang and N₁, N₂, N₃ and N₄ are different nucleotidesselected from G, A, T and C” refers to a population of sequences thatpotentially contain sequences 3′-N₁-5′ and 3′-N₂-5′ or optionally3′-N₃N₁-5′ and 3′-N₃N₂-5′ or 3′-N₃N₄N₁-5′ and 3′-N₃N₄N₂-5′ and,optionally, overhangs of sequence 3′-N₃N₄N₃N₁-5′ and 3′-N₃N₄N₃N₂-5′ andso on (e.g., overhangs of sequence 3′-N₃N₄N₃N₄N₁-5′ and 3′-N₃N₄N₃N₄N₂-5′and then 3′-N₃N₄N₃N₄N₃N₁-5′ and 3′-N₃N₄N₃N₄N₃N₂-5′).

As used herein, the term “alternating stretches” refers to twonucleotides stretches, where one “stretch” is a contiguous sequence of,e.g., up to 10, of the same nucleotide (e.g., a G, A, T or C), and thesecond stretch is contiguous sequence of, e.g., up to 10, of a differentnucleotide, that alternate with one another, i.e., one stretch (e.g., astring of T's) occupies the odd positions and the other stretch (e.g., astring of A's) occupies the even positions.

As used herein, the term “incomplete nucleotide mix” comprises anucleotide mix that contains one, two or three nucleotides (but not allfour nucleotides) selected from G, A, T and C. The nucleotides may belabeled or unlabeled.

As used herein, the term “reversible terminator” refers to a chemicallymodified nucleotide base that when incorporated into growing DNA strandby DNA polymerase blocks further incorporation of bases. Such“reversible terminator” base and DNA strand can be deprotected bychemical treatment and following such deprotection DNA strand can befurther extended by DNA polymerase.

As used herein, the term “fluorescently labeled reversible terminator”refers to a “reversible terminator” base which is labeled by fluorophorethrough linker cleavable by same treatment which is used to deprotectthe DNA strand which ends with this base. Deprotecting the“fluorescently labeled reversible terminator” simultaneously activatesthe DNA strand for further extension and removes the fluorescent labelfrom it.

For ease of description, many of the sequences described herein arewritten out in the 3′ to 5′ direction. While DNA sequences are routinelyset forth in 5′ to 3′ direction, for the ease description, certain DNAsequences in the text below are described in the 3′ to 5′ direction. Ineach such case the directionality is specifically annotated.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

In some embodiments the method comprises producing a labeled a planarsample (e.g., an FFPE section mounted on a planar surface such as amicroscope slide) using a capture agent that specifically binds tocomplementary sites in the planar sample. Methods for binding antibodiesand/or nucleic acids to sites in the planar sample are well known. Inthese embodiments, the capture agent in the labeled sample is linked toa double-stranded nucleic acid that comprises a first strand and asecond strand (e.g., two oligonucleotide that are hybridized together oran RCA product that is hybridized to oligonucleotides) and the captureagent is linked (covalently or non-covalently via a biotin) to thedouble-stranded nucleic acid by the first strand of the double-strandednucleic acid (e.g., by the 5′ end, the 3′ end, or anywhere in-between),and the 3′ end or 5′ end of one of the strands (e.g., the 3′ end of thefirst strand, any 3′ ends in the second strand, the 5′ end of the firststrand or any 5′ ends in the second strand) is extendible using theother strand as a template. In some cases, the 3′ end of the firststrand may be recessed relative to the 5′ end of the second strand,thereby defining an overhang. In other cases, the 5′ end of the firststrand may be recessed relative to the 3′ end of the second strand,thereby defining an overhang. In many embodiments, the capture agent iscross-linked the planar sample, thereby preventing the capture agentfrom disassociating during subsequent steps. This crosslinking step maybe done using any amine-to-amine crosslinker (e.g. formaldehyde,disuccinimiyllutarate or another reagents of similar action) although avariety of other chemistries can be used to cross-link the capture agentto the planar sample if desired. The method comprises reading afluorescent signal generated by addition of a nucleotide or shortoligonucleotide (e.g., of 2-10 bases) to the extendible end (e.g., the3′ end) of one of the strands. This step may be done by contacting theplanar sample with a polymerase and a nucleotide mix, a ligase and alabeled oligonucleotide, or a combination of the two, thereby adding oneor more nucleotides and/or a labeled oligonucleotide to the extendibleend; and reading a fluorescent signal generated by addition of the oneor more nucleotides or oligonucleotide to the extendible end.

As will be described in greater detail below, the fluorescent signal maybe generated by a variety of different methods. For example, in someembodiments, the fluorescent signal may be fluorescence from afluorescent nucleotide added to the end of the primer, or a FRET(fluorescence resonance energy transfer) signal resulting from the same.In other embodiments, the signal may generated by removing a quencherfrom a fluorescently labeled oligonucleotide that is also hybridized tothe oligonucleotide.

In any implementation of the method, the reading step may be followed byinactivating the fluorescence after reading so that other binding eventscan be detected and read. In these embodiments, the fluorescence may beinactivated by peroxide-based bleaching, cleavage of fluorophore linkedto nucleotide through cleavable linker (e.g. using TCEP as a cleavingreagent), base-exchange by exo+ polymerase such as Vent, or subsequentincorporation of quencher, for example.

Also, as will be described in greater detailed below, the method may bemultiplexed in a way that a single planar sample can be interrogated bya plurality of different capture agents, where each antibody is linkedto a different oligonucleotide (i.e., oligonucleotides of differentsequence). In multiplex embodiments, the planar sample may be labeledusing at least 5, at least 10, at least 20, at least 30, at least 50, orat least 100, up to 150 or more capture agents that are each linked to adifferent oligonucleotide, and binding of the capture agents can beseparately read using a fluorescence microscope equipped with anappropriate filter for each fluorophore, or by using dual or tripleband-pass filter sets to observe multiple fluorophores. See, e.g., U.S.Pat. No. 5,776,688. As noted below, the oligonucleotides linked to thecapture agent may act as a splint for a padlock probe, and as a primerfor initiating rolling circle amplification.

The capture agent used in some embodiments of the method may be linkedto a double-stranded oligonucleotide that contains a 5′ overhang (i.e.,a recessed 3′ end that can be extended by a polymerase or ligase) or a3′ overhang (i.e., a recessed 5′ end that can be extended by a ligase).An example of such a capture agent is shown in FIGS. 1 and 2. In theexample shown in FIG. 1B, the overhang is a single nucleotide overhang(e.g., an A), although a longer overhang (e.g., at least 2, at least 3,at least 4, at least 5, at least 6, at least 8, at least 10, at least20, or at least at least 30, may be useful for other applications (e.g.,multiplexed applications). As shown in FIG. 5 A-D, in certain cases, theoverhang may contain a repeated sequence, e.g., 2, 3, 4, 5, or 6 or morerepeats of the same sequence of 2, 3, 4, 5 or 6 nucleotides, therebyallowing the capture agent to be used in multiplexed applications asdescribed below. In certain embodiments, the double strandedoligonucleotide may have a recessed 3′ end at the other end of theoligonucleotide (i.e., at the end closest to the capture agent).However, this end may be designed to be not extendible. In certaincircumstances, the double-stranded oligonucleotide may contain one ormore third oligonucleotides that are hybridized to the overhang. Inthese embodiments, there will be a gap of 1, 2, 3, 4 or 5 or morenucleotides between the second strand of the double-strandedoligonucleotide and the oligonucleotide that is hybridized to theoverhang (see, e.g., FIGS. 7 and 8). In multiplex embodiments, theplurality of capture agents may be distinguished by the sequence of theoverhang and not by the sequence of the first strand of the doublestranded oligonucleotide. In these embodiments, the second strand of thedouble stranded oligonucleotides is different for each of the captureagents. As shown in other figures, the method may also be implementedusing capture agents that are linked to a primer that acts a splint forcircularlizing a padlock probe and for priming amplification ofcircularlized padlock probe by rolling circle amplification. In theseembodiments, the capture agents in the labeled sample may be linked to arolling circle amplification product.

In certain cases, the fluorophore used may be a coumarin, a cyanine, abenzofuran, a quinoline, a quinazolinone, an indole, a benzazole, aborapolyazaindacene and or a xanthene including fluorescein, rhodamineand rhodol. In multiplexing embodiments, fluorophores may be chosen sothat they are distinguishable, i.e., independently detectable, from oneanother, meaning that the labels can be independently detected andmeasured, even when the labels are mixed. In other words, the amounts oflabel present (e.g., the amount of fluorescence) for each of the labelsare separately determinable, even when the labels are co-located (e.g.,in the same tube or in the same area of the section).

Specific fluorescent dyes of interest include: xanthene dyes, e.g.,fluorescein and rhodamine dyes, such as fluorescein isothiocyanate(FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAMand F), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX),6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE or J),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G⁵ or G⁵),6-carboxyrhodamine-6G (R6G⁶ or G⁶), and rhodamine 110; cyanine dyes,e.g., Cy3, Cy5 and Cy7 dyes; coumarins, e.g., umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g., Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g., BODIPY dyes and quinoline dyes. Specificfluorophores of interest that are commonly used in subject applicationsinclude: Pyrene, Coumarin, Diethylaminocoumarin, FAM, FluoresceinChlorotriazinyl, Fluorescein, R110, Eosin, JOE, R6G,Tetramethylrhodamine, TAMRA, Lissamine, Napthofluorescein, Texas Red,Cy3, and Cy5, etc.

Suitable distinguishable fluorescent label pairs useful in the subjectmethods include Cy-3 and Cy-5 (Amersham Inc., Piscataway, N.J.), Quasar570 and Quasar 670 (Biosearch Technology, Novato Calif.), Alexafluor555and Alexafluor647 (Molecular Probes, Eugene, Oreg.), BODIPY V-1002 andBODIPY V1005 (Molecular Probes, Eugene, Oreg.), POPO-3 and TOTO-3(Molecular Probes, Eugene, Oreg.), and POPRO3 and TOPRO3 (MolecularProbes, Eugene, Oreg.). Further suitable distinguishable detectablelabels may be found in Kricka et al. (Ann Clin Biochem. 39:114-29,2002), Ried et al. (Proc. Natl. Acad. Sci. 1992: 89: 1388-1392) andTanke et al. (Eur. J. Hum. Genet. 1999 7:2-11) and others.

In addition to the labeling methods described above, the sample may bestained using a cytological stain, either before or after performing themethod described above. In these embodiments, the stain may be, forexample, phalloidin, gadodiamide, acridine orange, bismarck brown,barmine, Coomassie blue, bresyl violet, brystal violet, DAPI,hematoxylin, eosin, ethidium bromide, acid fuchsine, haematoxylin,hoechst stains, iodine, malachite green, methyl green, methylene blue,neutral red, Nile blue, Nile red, osmium tetroxide (formal name: osmiumtetraoxide), rhodamine, safranin, phosphotungstic acid, osmiumtetroxide, ruthenium tetroxide, ammonium molybdate, cadmium iodide,carbohydrazide, ferric chloride, hexamine, indium trichloride, lanthanumnitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid,phosphomolybdic acid, potassium ferricyanide, potassium ferrocyanide,ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate,thallium nitrate, thiosemicarbazide, uranyl acetate, uranyl nitrate,vanadyl sulfate, or any derivative thereof. The stain may be specificfor any feature of interest, such as a protein or class of proteins,phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle (e.g., cellmembrane, mitochondria, endoplasmic recticulum, golgi body, nulearenvelope, and so forth), a compartment of the cell (e.g., cytosol,nuclear fraction, and so forth). The stain may enhance contrast orimaging of intracellular or extracellular structures. In someembodiments, the sample may be stained with haematoxylin and eosin(H&E).

The structures of exemplary sulfhydryl-cleavable deoxynucleotideanalogues that can be used in the present method are shown below. Aswould be recognized, these nucleotides are only exemplary and othernucleotides, including nucleotides that are cleavable by other stimuli(e.g., photocleavable nucleotides) can be used in the present method.

In order to further illustrate the present invention, the followingspecific examples are given with the understanding that they are beingoffered to illustrate the present invention and should not be construedin any way as limiting its scope.

Implementation I

In this example, the fluorescent signal may be produced by a fluorescentnucleotide that is added to (i.e., added by a polymerase or, if thefluorescent nucleotide is in an oligonucleotide, ligated onto) the 3′end of the primer. This method may comprise reading a signal from theadded fluorescent nucleotide, or reading a FRET signal generated byenergy transfer between two fluorescent nucleotides that are added tothe primer.

The example shown in FIGS. 1 and 2 shows how an antibody can be linkedto a oligonucleotide chemically, or via biotin/streptavidin interactions(FIG. 1B) and how a fluorescent signal can be generated by adding afluorescent nucleotide to the end of the primer (FIG. 2). In thisexample, the antigen is stained by an antibody that is coupled to a DNAdimer with an overhanging 5′ end (lower strand) and recessed 3′ end(upper strand) either chemically (FIG. 1 top panel) or throughstreptavidin (FIG. 1 bottom and middle panels).

After binding the capture agent to the tissue sample, the pattern ofbinding of the capture agent may be determined using an on-slide endfill-in reaction by using a suitable polymerase (e.g., by exo⁻ Klenow,Bst, Taq, Klentaq, or an exo⁻ Klenow-Vent mixture) and fluorescentlylabeled nucleotide (FIG. 1 and FIG. 2 top panel).

If necessary, the signal-to-noise ratio can be increased by: a)multimerization of position complementary to labeling nucleotide (FIG.2, middle panel); or b) by generating a FRET between two nucleotides areincorporated, whereby the emission wavelength of one of the nucleotides(FIG. 2, bottom panel C on the figure) serves as an excitationwavelength for another (FIG. 2, bottom panel U on the figure).

Fluorescence may be inactivated before addition of subsequent stainingreagents by any convenient method including, but not limited tophotobleaching, peroxide-based bleaching, inactivation by ozone,cleavage of fluorophore linked to nucleotide through cleavable linker(e.g. using TCEP as a cleaving reagent), base-exchange by exo+polymerase such as Vent, subsequent incorporation of quencher.

In these embodiments, after fluorescence has been inactivated, themethod can be repeated, i.e., the planar sample may be re-stained usinga different antibody and fluorescence can be read.

Multiplexing

Multiplexing can be implemented using specially designedoligonucleotides using two different approaches, referred to as the“reversible terminator” and “missing base” approaches, which aredescribed in greater detail below. Both of these methods rely on acomposition comprising a plurality of (e.g., at least 5, at least 10, atleast 20, at least 30, at least 50, or at least 100, up to 150 or more)capture agents that recognize different complementary sites, wherein:each of the capture agents is linked to a double-stranded nucleic acid(e.g., oligonucleotide) that comprises a first strand and a secondstrand; the capture agents are linked to a double-stranded nucleic acidby the (e.g., the 5′ end of) the first strand; the 3′ end of one of thestrands in each of the double-stranded nucleic acids extendible usingthe other strand as a template, where the template is different for eachof the capture agents. Examples of such compositions are illustrated inFIGS. 3 and 4, where the template is an overhang. The general principleshown in FIGS. 3 and 4 can be extended to double stranded nucleic acidsthat comprise RCA products. FIG. 3 shows a population of capture agentsthat have a template (e.g., overhang) defined by the formula:3′-N_(4n)N₁/N₂/N₃-5′ followed by short stretch of random composition onthe 5′ end to increase the overall polymerase residence on the DNAduplex, where N₁, N₂, N₃ and N₄ are different nucleotides selected fromG, A, T and C and n is 0, 1 or more. FIG. 4, on the other hand, shows apopulation of capture agents that have an overhang defined by theformula 3′-YN₁/N₂-5′, optionally followed by short stretch (e.g., 1-5residues) of random nucleotides on the 5′ end to increase the overallpolymerase residence on the DNA duplex, wherein Y is a nucleotidesequence of length n (n is 0, 1 or more) composed of bases N₃ and N₄,wherein nucleotide N₃ is in odd positions and nucleotide N₄ is in evenpositions, counting from the start of the overhang and N₁, N₂, N₃ and N₄are different nucleotides selected from G, A, T and C. As illustrated inFIGS. 3, 4 and 5, the sequence of the first strand is the same for eachof the capture agents; and the sequence of the second strand isdifferent for each of the capture agents. In these embodiments, thedifferent second strands make the overhangs different between thedifferent capture agents.

In some embodiments, the multiplex methods may generally comprise: (a)incubating a planar sample with an above-described antibody compositionunder conditions by which the capture agents bind to complementary sitesin the planar sample; (b) cross-linking the capture agents to the planarsample; (c) contacting the planar sample with a polymerase and either anincomplete nucleotide mix of labeled and unlabeled nucleotides or anucleotide mix where some or all nucleotides are fluorescent and some orall nucleotides are reversible terminator nucleotides or fluorescentreversible terminator nucleotides and optionally, contacting the planarsample with a mixture of labelled and unlabeled oligonucleotides and aDNA ligase enzyme that covalently attaches the short labelledoligonucleotides to the 3′ end of the oligonucleotide duplexes that areattached to the specific capture agents. In these embodiments,oligonucleotides are only added to duplexes that an overhang that iscomplementary to the oligonucleotide. This method further comprises (d)reading, using fluorescence microscopy, a fluorescent signal generatedby addition a nucleotide to some but not all of the capture agents.Following signal registration, this method may comprise (e) removing thefluorescent signals by chemical or photocleavage of a labeled nucleotideif the reversible terminator approach is used, followed by deprotectingthe 3′ ends of the oligonucleotides, enabling the addition of furthernucleotides and/or oligonucleotides. Step (c) of this method maycomprise (c) contacting the planar sample with a polymerase and: (i) anucleotide mix that comprises fluorescent nucleotides that arecomplementary to N₁, N₂ and N₃ and a reversible terminator nucleotidethat is complementary to N₄ or (ii) a nucleotide mix that comprisesfluorescent reversible terminator nucleotides that are complementary toN₁, N₂ and N₃ and a reversible terminator nucleotide that iscomplementary to N₄ or (iii) a nucleotide mix that comprises fluorescentnucleotides that are complementary to N₁, and N₂, an unlabelednucleotide that is complementary to N₃, and no nucleotide that iscomplementary to N₄, thereby adding fluorescent nucleotides onto thedouble-stranded oligonucleotides of some but not all of the captureagents thereby adding fluorescent nucleotides onto the double-strandedoligonucleotides of some but not all of the capture agents; and (d)reading, using fluorescence microscopy, a fluorescent signal generatedby addition of a fluorescent nucleotide to some but not all of thecapture agents. Step (c) can also be implemented by adding a labeledoligonucleotide to the duplex using a ligase. Examples of such methodsare described in greater detail below.

With reference to FIG. 6 it is expected that in the case when largerpanels of capture agents are to be employed (e.g. 100 and more) thelength of the read over the oligonucleotide overhangs may increaseaccordingly. This may or may not reduce the efficiency of staining dueto accumulation of primer extension errors along the length of theoligonucleotide duplex. To circumvent such potential source of signalloss a slight modification of design can be implemented. The pluralityof capture agents can be divided in sets such that number of captureagents in the set does exceed the capacity of the multiplexing protocolto render staining without significant signal loss (e.g. 30). Each suchset of capture agents will be conjugated to “terminated” (the last 3′base is dideoxy- or propyl-modified) upper strand oligonucleotide of thesame sequence as in the original version of the “missing base” approach.The lower strand oligonucleotides will incorporate an additionalset-specific region which will serve as a landing spot for an additionalprimer which is to be on-slide hybridized to the particular subset ofthe total plurality of the antibodies at the time when they are to berendered. This approach allows not to extend the reads beyond certainthreshold and at the same time have an unlimited potential number ofcapture agents in the sample.

Reversible Terminator Method

This implementation of the method relies on reversible terminators,i.e., chain terminator nucleotides that can be de-protected afterincorporation, thereby allowing further nucleotides to be added to thatnucleotide.

This method can be implemented using a composition comprising aplurality of capture agents that are linked to a double stranded nucleicacid (e.g., oligonucleotides), as illustrated in FIG. 3. In theseembodiments, the top strand of the double stranded nucleic acid islinked to the capture agent and may be the same for each antibody, andthe sequence of the bottom strand varies between capture agents. Asshown on FIG. 5A, the 5′ end of the lower strand of the double-strandednucleic acid (which may form an overhang) is of the general3′-N_(4n)N₁/N₂/N₃-5′ followed by short stretch of random nucleotides onthe 5′ end to increase the overall polymerase residence on the DNAduplex, where N₁, N₂, N₃ and N₄ are different nucleotides selected fromG, A, T and C and n is 0, 1 or more. As shown on FIG. 5B a more generalformula of lower oligonucleotide overhang 3′-XN₁/N₂/N₃-5′, where N₁, N₂,N₃ are different nucleotides selected from G, A, T and C and X is anucleotide stretch of bases Xi (such that Xi are different nucleotidesselected from G, A, T and C) of random composition and length is alsoapplicable in this method.

In certain embodiments, this method may comprise: (a) incubating aplanar sample with a multiplex antibody composition in which theoverhangs are of the formula 5′-N₁/N₂/N₃N_(4n), wherein N₁, N₂, N₃ andN₄ are different nucleotides selected from G, A, T and C and n is 1 ormore; under conditions by which the capture agents specifically bind tocomplementary sites in the planar sample; (b) cross-linking the captureagent to the planar sample; (c) contacting the planar sample with apolymerase and a nucleotide mix that comprises fluorescent nucleotidesthat are complementary to N₁, N₂ and N₃ and a reversible terminatornucleotide that is complementary to N₄ and/or ligating a oligonucleotidethat comprises a labeled nucleotide; and (d) reading, using fluorescencemicroscopy, a fluorescent signal generated by addition of a nucleotideto some but not all of the capture agents. This cycle may be repeated by(e) inactivating the fluorescent signal, deprotecting the reversibleterminator nucleotide and (f) blocking the planar sample; and repeatingsteps (c) and (d). In certain embodiments, the method may compriserepeating steps (c), (d) (e) and (f) multiple times. The reagent usedfor blocking may vary depending on the chemistry used. In certainembodiments, the sample may be blocked with a thiol-reactive compoundssuch as cysteine, glutathione or iodoacetamide.

For example, this method can be implemented using a compositioncomprising: a first antibody linked to a first double strandedoligonucleotide, wherein the first double stranded oligonucleotidecomprises a single nucleotide 5′ overhang comprising base N₁; a secondantibody linked to a second double stranded oligonucleotide, wherein thesecond double stranded oligonucleotide comprises a single nucleotide 5′overhang comprising base N₂; a third antibody linked to a third doublestranded oligonucleotide, wherein the third double strandedoligonucleotide comprises a single nucleotide 5′ overhang comprisingbase N₃; a fourth antibody linked to a fourth double strandedoligonucleotide comprises a two nucleotide 5′ overhang, wherein thefirst position of the overhang comprises base N₄ and the second positionof the overhang is base N₁; a fifth antibody linked to a fifth doublestranded oligonucleotide, wherein the fifth double strandedoligonucleotide comprises a two nucleotide 5′ overhang, wherein thefirst position of the overhang comprises base N₄ and the second positionof the overhang is base N₂; and a sixth antibody linked to a sixthdouble stranded oligonucleotide, wherein the sixth double strandedoligonucleotide comprises a two nucleotide 5′ overhang, wherein thefirst position of the overhang comprises base N₄ and the second positionof the overhang is base N₃, wherein N₁, N₂, N₃ and N₄ are differentnucleotides selected from G, A, T and C. An example of such a populationof capture agents is shown in FIG. 3.

In RCA embodiments, the strand linked to the antibodies may be differentfor each of the antibodies, where the RCA product contains a sequenceconforming to the formula described above in each repeat of the RCAproduct.

In certain implementations, the composition may also contain a seventhantibody linked to a seventh double stranded oligonucleotide, whereinthe seventh double stranded oligonucleotide comprises a multiplenucleotide 5′ overhang, wherein the first position of the overhangcomprises base N₄, the second position of the overhang is base N₄ andthird is selected from N₁, N₂, and N₃. The same principle may be appliedto overhangs that have more than 7 positions (e.g., 9, 10, 11 up to 20,30, or 40 ore more) positions.

In this implementation of the method, the planar sample can beco-stained simultaneously using a panel of capture agents, each labeledwith one oligonucleotide duplex designed according to the strategyoutlined on FIG. 3. The duplexes are designed in such a way that eachantibody has the same upper strand sequence linked, covalently orthrough streptavidin, to an antibody through the 5′ end. The lowerstrand changes from antibody to antibody. In this implementation, thegeneral formula for the lower strand is3′-dideoxydC-sequence-complimentary-to-upper-strand G_(n)A/T/C-5′. Onetype of lower strand base (nucleotide G in this example) is reserved forstep-wise progression and its complementary pair on the upper strand isnever used in labeled form. The other three bases are complementary tolabeled nucleotides and can be used to identify three capture agents percycle. In a more general case the general formula for the lower strandis 3′-dideoxydC-sequence-complimentary-to-upper-strand-X—N₁/N₂/N₃-5′where X_(i) of X is any nucleotide excluding one reserved for “walkingbase” of this particular cycle and X is any base as shown on FIG. 5B.This design ensures that: a) no two antibody species contain the sameduplex and b) only three different capture agents are detected at atime. Each cycle includes: (a) a labeling step in which the threecapture agents are labeled and duplexes on the rest are extended onebase at a time, (b) an imaging step and (c) a destaining/deprotectionstep. During cycle to cycle transition the added fluorescent labels fromthe previous cycle are inactivated by any of the suitable methods,including but not limited to: cleavage of fluorophore off the nucleotide(if the labeled nucleotide is linked to the fluorophore through acleavable linker); peroxide based bleaching; photobleaching;chemically-assisted photobleaching; labeled base replacement by exo+polymerase, etc. After or simultaneously with inactivation of thefluorophores added in the previous reaction, the unlabeled “extension”nucleotide that has been added to the remainder of the capture agents isactivated by cleavage of the protective group off its 3′ end. Cleavageof the protective group, in turn, allows that nucleotide to be extendedin the next cycle. Since the A, T and C are reserved for incorporationof a labelled nucleotide, those nucleotides only occur at the end ofeach lower strand of the duplex. This approach is based on the chemicalnature of reversible terminators, which precludes upper strand extensionfor more than one nucleotide at a time even on polyG stretches of thelower strand. Optionally, a quencher labeled nucleotide can beincorporated following the labeled nucleotide. The performance of“reversible terminator method” as exemplified in sequential detection ofCD4 and CD8 positive T-cells in smears of mouse splenocytes isillustrated in FIG. 13A-D.

Missing Base Method

This implementation of the method relies on a “missing” base design inwhich, in each cycle, two labeled and one unlabeled nucleotides areadded to the reaction, and the “missing base” prevents the primers frombeing extended by more than a single nucleotide.

This method can be implemented using a composition comprising aplurality of capture agents that are linked to double stranded nucleicacids, as illustrated in FIG. 4. In these embodiments, the top strand ofthe double stranded nucleic acids is linked to the capture agent and maybe the same for each antibody, and the sequence of the bottom strandvaries between capture agents. As shown in FIG. 4, the 5′ end of thelower strand of the double-stranded oligonucleotide (which forms theoverhang) is of the general formula 3′-YN₁/N₂-5′, optionally followed byshort stretch (e.g., 1-5 residues) of random nucleotides on the 5′ endto increase the overall polymerase residence on the DNA duplex, whereinY is a nucleotide sequence of length n (n is 0, 1 or more) composed ofbases N₃ and N₄, wherein nucleotide N₃ is in odd positions andnucleotide N₄ is in even positions, counting from the start of theoverhang and N₁, N₂, N₃ and N₄ are different nucleotides selected fromG, A, T and C.

Also a more general formula 3′-YN₁/N₂-5′, wherein N₁, N₂, N₃ and N₄ aredifferent nucleotides selected from G, A, T and C and Y is a nucleotidesequence of length n (n is 0, 1 or more) composed of alternating randomlength stretches of bases N₃ and N₄ such that the order number ofN₃—stretches is odd and of N₄ stretches is even, may be applicable inthis method

In certain embodiments, this method may comprise: (a) incubating aplanar sample with a multiplex antibody composition in which theoverhangs are of the formula (3′-YN₁/N₂-5′) described in the priorparagraph; under conditions by which the capture agents specificallybind complementary sites in the planar sample; (b) cross-linking thecapture agent to the planar sample; (c) contacting the planar samplewith a polymerase and a nucleotide mix that comprises fluorescentnucleotides that are complementary to N₁, and N₂, an unlabelednucleotide that is complementary to N₃ and no nucleotide that iscomplementary to N₄ and/or ligating an oligonucleotide that has alabeled nucleotide; and (d) reading, using fluorescence microscopy, afluorescent signal generated by addition of a nucleotide to some but notall of the capture agents. This cycle may be repeated by (e)inactivating the fluorescent signal, (f) blocking the sample andcontacting the planar sample with a polymerase and an unlabelednucleotide that is complementary to N₄ and/or contacting the sample witha labeled oligonucleotide and a ligase; and repeating steps (c) (d). Incertain embodiments, the method may comprise repeating steps (c), (d),(e) and (f) multiple times.

This method can be implemented using a capture agent composition thatcomprises: a first antibody linked to a first double strandedoligonucleotide, wherein the first double stranded oligonucleotidecomprises a single nucleotide 5′ overhang comprising base N₁; a secondantibody linked to a second double stranded oligonucleotide, wherein thesecond double stranded oligonucleotide comprises a single nucleotide 5′overhang comprising base N₂; a third antibody linked to a fourth doublestranded oligonucleotide, wherein the third double strandedoligonucleotide comprises a two nucleotide 5′ overhang, wherein thefirst from the 3′ position of the overhang comprises base N₄ and thesecond position comprises N₁; and a fourth antibody linked to a fourthdouble stranded oligonucleotide, wherein the fourth double strandedoligonucleotide comprises a two nucleotide 5′ overhang, wherein thefirst position of the overhang comprises base N₄ and the second positioncomprises base N₂, wherein N₁, N₂, N₃ and N₄ are different nucleotidesselected from G, A, T and C. An example of such a population of captureagents is shown in FIG. 4.

In certain implementations, the composition may also contain a fifthantibody linked to a fifth double stranded oligonucleotide, wherein thefifth double stranded oligonucleotide comprises a multiple nucleotide 5′overhang, wherein the first position of the overhang comprises base N₄,the second position comprises base N₃, and the third position comprisesN₁ or N₂.

Overall there is no theoretical limits to the number of co-detectedcomplementary sites, e.g., antigens, both in the case of “reversibleterminator” and of “missing base” approach

The missing base approach does not use reversible terminators. Instead,extension of a single nucleotide is ensured by using two interchangingbases (e.g., T and C as shown in FIG. 4 instead of the corresponding Gin the “reversible terminators” approach) and adding only one of the twodNTPs at a time in the primer extension reaction. After theincorporation of the first nucleotide, the absence of the second dNTPcauses strand elongation to stall, thereby ensuring that the primers areextended by only a single nucleotide. As in the previous strategy, allcomplementary sites can be co-stained simultaneously using captureagents, each labeled with a specific oligonucleotide duplex.

In this embodiment, the duplexes can be designed using the strategyshown in FIG. 4, i.e., in such a way that each antibody has the sameupper stand oligonucleotide sequence linked to it via covalent bond orthrough a streptavidin-biotin interaction. In this implementation, thelower strand changes from antibody to antibody. In this method, thegeneral formula for the lower strand is 3′ddC-sequence-complimentary-to-upper-strand-YA/N₂-5′ where Y is composedof bases T and C such that T can be found only in even and C only at oddpositions. Or in the more general case 3′-YN₁/N₂-5′, wherein N₁, N₂, N₃and N₄ are different nucleotides selected from G, A, T and C and Y is anucleotide sequence of length n (n is 0, 1 or more) composed ofalternating random length stretches of bases N₃ and N₄ such that theorder number of N₃—stretches is odd and of N₄ stretches is even. In thefirst simple implementation two base pairs of the lower strand (T and Cas in exemplary design on FIG. 4) are reserved for step-wise progressionand their complementary pair on the upper strand is never labeled. Theother two bases are complementary to labeled nucleotides and can renderthe staining with two different capture agents per cycle. Such designensures that a) no two capture agents contain the same duplex and b)only two different antibody are read per cycle. In this implementation,each cycle can have three steps: a labeling step in which the twocapture agents are labeled by incorporation of fluorescent dNTPs and allof the other duplexes are extended one base at a time, an imaging step,and a de-staining/reactivation step.

In RCA embodiments, the strand linked to the antibodies may be differentfor each of the antibodies, where the RCA product contains a sequenceconforming to the formula described above in each repeat of the RCAproduct.

During cycle-to-cycle transition the labeled capture agents from theprior cycle can be bleached/destained in the same way as describedabove. Optionally, instead of bleaching, a quencher labeled nucleotidecan be incorporated after the labeled base. Because, in this embodiment,the position that is labeled is the last position in the overhang, thelabeled capture agents from prior cycle cannot be re-labeled in latercycles because all nucleotide positions in the overhang have been filledin. The performance of “reversible terminator method” as exemplified insequential detection of CD4 and CD8 positive T-cells in smears of mousesplenocytes is illustrated in FIG. 13, 15 and FIG. 16.

Implementation II

In this method, extension of a primer by nick translation removes aquencher from a fluorescently labeled “detector” oligonucleotide that ishybridized to the lower strand oligonucleotide in such a way that ispositioned downstream from the upper strand primer. The principles ofthis method are illustrated in FIG. 7. A multiplexed version of thismethod is shown in FIG. 8.

In certain embodiments, the multiplexed implementations may comprise:(a) incubating the planar sample with a plurality of capture agents thatare linked to a double-stranded oligonucleotide; (b) crosslinking thecapture agents to the planar sample; (c) extending a primer that ishybridized to the oligonucleotide of a first set of capture agents ofthe plurality, thereby generating a first set of fluorescent signals(e.g., by removing the quencher from a labeled oligonucleotide that ishybridized to the oligonucleotide downstream from the primer), e.g., byadding a nucleotide using a polymerase or by adding an oligonucleotideusing a ligase; (d) reading the first set of fluorescent signals usingfluorescence microscopy; (e) inactivating the fluorescence; (f)extending a primer that is hybridized to the oligonucleotide of a secondset of capture agents of the plurality, thereby generating a second setof fluorescent signals (e.g., by removing the quencher from a labeledoligonucleotide that is hybridized to the oligonucleotide downstreamfrom the primer); (g) reading the second set of fluorescent signalsusing fluorescence microscopy; and (h) comparing the images produce insteps (d) and (g).

In this method, the architecture of the double-stranded oligonucleotideslinked to the capture agent has a specific design which is effectivelyenabling rendering of the capture agent binding pattern by “nicktranslation”. In particular the duplex of the upper strand and the lowerstrand oligonucleotide with long 5′ overhang of the lower strand isfurther hybridized to a small detector oligonucleotide labeled both byfluorescent and the quencher. There is a predesigned gap between theinitial upper strand and the upper strand detector oligo. During cyclicstaining this gap is “walked” by either “reversible terminator” or“missing base” (similar to described in previous sections) until the gapis reduced to a single base nick. Extension and progression through thenick on the upper strand by “nick translating” polymerase such as DNApol I removes the quencher from some but not all of the quenchedfluorescently labeled oligonucleotides, thereby generating a fluorescentsignal for some but not all of the capture agents.

In some embodiments the method generally comprises: (i) labeling aplanar sample with: i. a first antibody, wherein the first antibody islinked to a first oligonucleotide duplex comprising, lower strandoligonucleotide with a unique sequence hybridized thereto: (i) anoligonucleotide upper strand “primer” and (ii) a labeled upper strandoligonucleotide comprising a 5′ quencher at a site that is downstreamfrom the primer; and a fluorophore downstream from the quencher and ii.a second antibody, wherein the second antibody is linked to a secondoligonucleotide duplex comprising, lower strand oligonucleotide withunique sequence hybridized thereto: (i) an oligonucleotide upper strand“primer” and (ii) an upper strand oligonucleotide labeled both byfluorophore and a quencher; wherein the gap between the 3′ end of theprimer and the 5′ end of the labeled oligonucleotide is different forthe first and second oligonucleotides; (ii) incubating the tissue samplewith a first nucleotide mix and a polymerase, thereby removing thequencher from only the labeled oligonucleotide that is hybridized to thefirst oligonucleotide and producing a first fluorescent signal; (iii)reading the first fluorescent signal using fluorescence microscopy; (iv)inactivating the fluorescent signal by further progression ofnick-translating polymerase; (v) incubating the tissue sample with asecond nucleotide mix and a polymerase, thereby removing the quencherfrom only the labeled oligonucleotide that is hybridized to the secondoligonucleotide and producing a first fluorescent signal; and (vi)reading the second fluorescent signal from the planar sample usingfluorescence microscopy.

FIGS. 7 and 8 show an example of this method. The multiplexing methodshown in FIG. 8 has the following steps:

Step 1: The planar sample is stained by capture agents that are coupledto a DNA double-stranded oligonucleotide chemically or throughstreptavidin (as described in FIG. 1) such that the top strand of theduplex contains a nick or a single base deletion followed by anucleotide stretch bordered by a fluorophore and its quencher on twoends (“molecular beacon” or Taqman based design).

Step 2: Staining pattern is rendered by a nick-translation reactioncarried out by any 5′ exo+ polymerase such as DnaPoll Klenow fragment inthe presence of a single letter (A as in FIG. 5 for example). Nicktranslation removes the quencher but stops before removing the part ofthe duplex with the fluorophore.

Step 3: For rendering of other staining reagents, the fluorescence isremoved by continuing nick translation in the presence of the letters ofthe stretch bearing the fluorophore.

Step 4: When multiplexing is desired, multiplexing can be achieved byspecial design of oligonucleotide duplexes attached to detectionreagents. In particular each antibody set (two or three per cycle) has agap of an increasing length between the top strand priming and thedetector oligonucleotide. This sequence gap on the strand bearing thequencher/fluorophore pair is filled up to final nick in such a way thatsingle base is extended per cycle, similar to how it is achieved inmethod 1 (see FIG. 8).

Implementation III

In this implementation, the method comprises rending antibody stainingby primer extension with a fluorophore labeled base or otherwise readinga FRET signal generated by energy transfer between a first fluorescentnucleotide added to the primer by primer extension and a secondnucleotide that is present in the oligonucleotide FIG. 10. Theprinciples of this method are illustrated in FIG. 9A. The multiplexingis achieved by removing the extension priming oligonucleotide by meltingthe duplex or by exonuclease and reannealing another primeroligonucleotide which is extendable on a different antibody. Amultiplexed version of this method is shown in FIG. 9B. In certainembodiments, the multiplexed implementations may comprise: (a)incubating the planar sample with a plurality of capture agents; (b)cross-linking the capture agents to the planar sample; (c) extending aprimer that is hybridized to the oligonucleotide of a first set ofcapture agents of the plurality (e.g., wherein the 3′ end of the firstprimer anneals to only the oligonucleotide of the first population),thereby generating a first set of fluorescent signals (which step can bedone by adding a labeled nucleotide using polymerase and/or contactingthe sample with a labeled oligonucleotide and a ligase); (d) reading thefirst set of fluorescent signals using fluorescence microscopy; (e)inactivating the fluorescence; (f) extending a primer that is hybridizedto the oligonucleotide of a second set of capture agents of theplurality (e.g., wherein the 3′ end of the first primer anneals to onlythe oligonucleotide of the second population), thereby generating asecond set of fluorescent signals (which step can also be done by addinga labeled nucleotide using polymerase and/or contacting the sample witha labeled oligonucleotide and a ligase); (g) reading the second set offluorescent signals using fluorescence microscopy; and (h) comparing theimages produce in steps (d) and (g).

In certain embodiments, this method comprises: (a) incubating the planarsample with (i) a first antibody that is linked to a first labeledoligonucleotide and (ii) a second antibody that is linked to a secondlabeled oligonucleotide, (b) cross-linking the capture agents to theplanar sample; (c) hybridizing the first and second labeledoligonucleotides with a first primer, wherein the 3′ end of the firstprimer anneals to only the first labeled oligonucleotide; (d) extendingthe primer with a fluorescent nucleotide (which step can be done byadding a labeled nucleotide using polymerase and/or contacting thesample with a labeled oligonucleotide and a ligase); (e) reading, byfluorescence microscopy, a FRET signal generated by energy transferbetween the label of the first oligonucleotide and the fluorescentnucleotide added to the first primer; (f) inactivating the fluorescentnucleotide added to the first primer; (g) hybridizing the first andsecond labeled oligonucleotides with a second primer, wherein the 3′ endof the second primer anneals to only the second labeled oligonucleotide;(h) extending the second primer with a fluorescent nucleotide; and (i)reading, by fluorescence microscopy, a FRET signal generated by energytransfer between the label of the second oligonucleotide and thefluorescent nucleotide added to the second primer.

FIGS. 9-10 shows an example of this method. The method shown in FIGS.8-11 has the following steps:

Step 1: The planar sample is stained using a capture agent that iscoupled to a single stranded oligonucleotide. The oligonucleotide couldbe either unlabeled or labeled by FRET acceptor (e.g. Cy5) fluorophoreon the 3′ end.

Step 2: The binding pattern can be determined by an on-slidehybridization of a complementary probe followed a primer extensionreaction in which a fluorescently labeled nucleotide fills in theoverhang in the extended strand. In this example (see FIG. 10) theextended base is labeled by a FRET donor (e.g. Cy3), which can increasethe signal to noise ratio. If the oligonucleotide that is linked to thecapture agent is unlabeled, then the fluorescent emission of thenucleotide that has been incorporated by DNA synthesis can be detecteddirectly, without FRET FIG. 9.

Step 3: The binding pattern of other capture agents can be determined byremoving the fluorescence by cleavage of lower strand by exo+ DNApolymerase such as Vent (FIG. 9). Alternatively, the fluorescence can beremoved by raising the temperature beyond the melting point of the DNAstrands or by one of the de-staining techniques described previously.

Step 4: Multiplexing can be achieved by staining of the sample with alibrary of capture agents each labeled with specific oligonucleotidesand cycling through Steps 1-3, as described above, each time using adifferent detection oligonucleotide that is complementary to one of thecapture agent-conjugated oligonucleotides. Only duplexes where primersare annealed specifically will be properly extended (FIG. 11). In theseembodiments, each primers is designed so that its 3′ end hybridizes toonly one of the oligonucleotides that are linked to a capture agent.

Further Implementations

As schematically illustrated in FIG. 16, the signal may be amplifiedusing rolling circle amplification. In these embodiments, a captureagent that is linked to an oligonucleotide is hybridized to a padlockprobe that hybridizes to the oligonucleotide in such a way that the endsof the padlock probe are ligatably adjacent. In this embodiment, afterligation, the padlock probe (which is now circularized) can be copied bya rolling circle amplification reaction that is primed by theoligonucleotide. This reaction results in a concatamer of the padlockprobe that contains several (in many cases hundreds or thousands) ofcopies of the same sequence in tandem that is linked to the captureagent. The rolling circle amplification product (which is linked to theantibody) can be detected using methods described above and, asillustrated, the signal is amplified because the sequence being detectedis repeated. In these embodiments, the (i) the capture agent is linkedto a double-stranded nucleic acid that comprises a first strand (i.e.,the RCA product) and a second strand (comprising the detectionoligonucleotides). Single molecules can be detected using such methods.

FIG. 19 shows how RNA molecules can be detected using a padlockprobe/RCA amplification approach. In this method, the padlock probehybridizes to the same mRNA as the capture agent (the “splint-primer”),thereby ensuring that the padlock probe circularizes only in thepresence of the target RNA. In this embodiment, the splint-primerhybridizes to the target RNA, acts as a splint for the padlock probe,and also acts as a primer for rolling circle amplification, therebyallowing the signal to be amplified in a similar to FIG. 16.

FIG. 20 shows an alternative method that relies on primer extension andthe ligation of a short, labeled oligonucleotide. In this embodiment,ligation of short labeled oligonucleotide to the top strandoligonucleotide only occurs after the overhang has been filed in to acertain point. In embodiments that rely on ligation, a labeledoligonucleotide can be added to either the 3′ end or the 5′ end of theextendible end.

Utility

The methods and compositions described herein find general use in a widevariety of application for analysis of any planar sample (e.g., in theanalysis of tissue sections, sheets of cells, spun-down cells, blots ofelectrophoresis gels, Western blots, dot-blots, ELISAs, antibodymicroarrays, nucleic acid microarrays etc).

In particular embodiments, the planar sample may be a section of atissue biopsy obtained from a patient. Biopsies of interest include bothtumor and non-neoplastic biopsies of skin (melanomas, carcinomas, etc.),soft tissue, bone, breast, colon, liver, kidney, adrenal,gastrointestinal, pancreatic, gall bladder, salivary gland, cervical,ovary, uterus, testis, prostate, lung, thymus, thyroid, parathyroid,pituitary (adenomas, etc.), brain, spinal cord, ocular, nerve, andskeletal muscle, etc.

In certain embodiments, capture agents specifically bind to biomarkers,including cancer biomarkers, that may be proteinaceous or a nucleicacid. Exemplary cancer biomarkers, include, but are not limited tocarcinoembryonic antigen (for identification of adenocarcinomas),cytokeratins (for identification of carcinomas but may also be expressedin some sarcomas), CD15 and CD30 (for Hodgkin's disease), alphafetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD117(for gastrointestinal stromal tumors), CD10 (for renal cell carcinomaand acute lymphoblastic leukemia), prostate specific antigen (forprostate cancer), estrogens and progesterone (for tumouridentification), CD20 (for identification of B-cell lymphomas) and CD3(for identification of T-cell lymphomas).

The above-described method can be used to analyze cells from a subjectto determine, for example, whether the cell is normal or not or todetermine whether the cells are responding to a treatment. In oneembodiment, the method may be employed to determine the degree ofdysplasia in cancer cells. In these embodiments, the cells may be asample from a multicellular organism. A biological sample may beisolated from an individual, e.g., from a soft tissue. In particularcases, the method may be used to distinguish different types of cancercells in FFPE samples.

The method described above finds particular utility in examining planarsamples using a plurality of antibodies, each antibodies recognizing adifferent marker. Examples of cancers, and biomarkers that can be usedto identify those cancers, are shown below. In these embodiments, onedoes not need to examine all of the markers listed below in order tomake a diagnosis.

Acute Leukemia IHC Panel CD3, CD7, CD20, CD34, CD45, CD56, CD117, MPO,PAX-5, and TdT. Adenocarcinoma vs. Mesothelioma IHC Pan-CK, CEA, MOC-31,BerEP4, TTF1, calretinin, and WT-1. Panel Bladder vs. Prostate CarcinomaIHC Panel CK7, CK20, PSA, CK 903, and p63. Breast IHC Panel ER, PR,Ki-67, and HER2. Reflex to HER2 FISH after HER2 IHC is available.Burkitt vs. DLBC Lymphoma IHC panel BCL-2, c-MYC, Ki-67. CarcinomaUnknown Primary Site, Female CK7, CK20, mammaglobin, ER, TTF1, CEA,CA19-9, S100, (CUPS IHC Panel - Female) synaptophysin, and WT-1.Carcinoma Unknown Primary Site, Male CK7, CK20, TTF1, PSA, CEA, CA19-9,S100, and (CUPS IHC Panel - Male) synaptophysin. GIST IHC Panel CD117,DOG-1, CD34, and desmin. Hepatoma/Cholangio vs. Metastatic HSA (HepPar1), CDX2, CK7, CK20, CAM 5.2, TTF-1, and Carcinoma IHC Panel CEA(polyclonal). Hodgkin vs. NHL IHC Panel BOB-1, BCL-6, CD3, CD10, CD15,CD20, CD30, CD45 LCA, CD79a, MUM1, OCT-2, PAX-5, and EBER ISH. LungCancer IHC Panel chromogranin A, synaptophysin, CK7, p63, and TTF-1.Lung vs. Metastatic Breast Carcinoma IHC TTF1, mammaglobin, GCDFP-15(BRST-2), and ER. Panel Lymphoma Phenotype IHC Panel BCL-2, BCL-6, CD3,CD4, CD5, CD7, CD8, CD10, CD15, CD20, CD30, CD79a, CD138, cyclin D1,Ki67, MUM1, PAX- 5, TdT, and EBER ISH. Lymphoma vs. Carcinoma IHC PanelCD30, CD45, CD68, CD117, pan-keratin, MPO, S100, and synaptophysin.Lymphoma vs. Reactive Hyperplasia IHC BCL-2, BCL-6, CD3, CD5, CD10,CD20, CD23, CD43, cyclin Panel D1, and Ki-67. Melanoma vs. Squamous CellCarcinoma CD68, Factor XIIIa, CEA (polyclonal), S-100, melanoma IHCPanel cocktail (HMB-45, MART-1/Melan-A, tyrosinase) and Pan- CK.Mismatch Repair Proteins IHC Panel MLH1, MSH2, MSH6, and PMS2.(MMR/Colon Cancer) Neuroendocrine Neoplasm IHC Panel CD56,synaptophysin, chromogranin A, TTF-1, Pan-CK, and CEA (polyclonal).Plasma Cell Neoplasm IHC Panel CD19, CD20, CD38, CD43, CD56, CD79a,CD138, cyclin D1, EMA, kappa, lambda, and MUM1. Prostate vs. ColonCarcinoma IHC Panel CDX2, CK 20, CEA (monoclonal), CA19-9, PLAP, CK 7,and PSA. Soft Tissue Tumor IHC Panel Pan-CK, SMA, desmin, S100, CD34,vimentin, and CD68. T-Cell Lymphoma IHC panel ALK1, CD2, CD3, CD4, CD5,CD7, CD8, CD10, CD20, CD21, CD30, CD56, TdT, and EBER ISH. T-LGLLeukemia IHC panel CD3, CD8, granzyme B, and TIA-1. UndifferentiatedTumor IHC Panel Pan-CK, S100, CD45, and vimentin.

In some embodiments, the method may be employed to detect the locationand, optionally, the abundance of DNA molecules and/or RNA molecules insitu. In one exemplary embodiment, the method may be used to detectintracellular RNAs. In these embodiments, the capture agent may be anucleic acid, and the intracellular location and, optionally, theabundance of RNA molecules (e.g., mRNAs or lncRNAs) may be detected insitu. Such hybridization methods may be adapted from known RNA or DNAFISH methods (see, e.g., Mahadevaiah et al (Methods Mol Biol. 2009558:433-44), Shaffer et al (PLoS One. 2013 8:e75120) and Pollex et al(Methods Mol. Biol. 2013 1042:13-31), which are incorporated byreference herein.

In some embodiments, the method may involve obtaining an image asdescribed above (an electronic form of which may have been forwardedfrom a remote location) and may be analyzed by a doctor or other medicalprofessional to determine whether a patient has abnormal cells (e.g.,cancerous cells) or which type of abnormal cells are present. The imagemay be used as a diagnostic to determine whether the subject has adisease or condition, e.g., a cancer. In certain embodiments, the methodmay be used to determine the stage of a cancer, to identify metastasizedcells, or to monitor a patient's response to a treatment, for example.

The compositions and methods described herein can be used to diagnose apatient with a disease. In some cases, the presence or absence of abiomarker in the patient's sample can indicate that the patient has aparticular disease (e.g., cancer). In some cases, a patient can bediagnosed with a disease by comparing a sample from the patient with asample from a healthy control. In this example, a level of a biomarker,relative to the control, can be measured. A difference in the level of abiomarker in the patient's sample relative to the control can beindicative of disease. In some cases, one or more biomarkers areanalyzed in order to diagnose a patient with a disease. The compositionsand methods of the disclosure are particularly suited to identifying thepresence or absence of, or determining expression levels, of a pluralityof biomarkers in a sample.

In some cases, the compositions and methods herein can be used todetermine a treatment plan for a patient. The presence or absence of abiomarker may indicate that a patient is responsive to or refractory toa particular therapy. For example, a presence or absence of one or morebiomarkers may indicate that a disease is refractory to a specifictherapy and an alternative therapy can be administered. In some cases, apatient is currently receiving the therapy and the presence or absenceof one or more biomarkers may indicate that the therapy is no longereffective.

In any embodiment, data can be forwarded to a “remote location”, where“remote location,” means a location other than the location at which theimage is examined. For example, a remote location could be anotherlocation (e.g., office, lab, etc.) in the same city, another location ina different city, another location in a different state, anotherlocation in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems can be in the same room but separated, or at least in differentrooms or different buildings, and can be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” informationreferences transmitting the data representing that information aselectrical signals over a suitable communication channel (e.g., aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. Examples of communicatingmedia include radio or infra-red transmission channels as well as anetwork connection to another computer or networked device, and theinternet or including email transmissions and information recorded onwebsites and the like. In certain embodiments, the image may be analyzedby an MD or other qualified medical professional, and a report based onthe results of the analysis of the image may be forwarded to the patientfrom which the sample was obtained.

In some cases, the method may be employed in a variety of diagnostic,drug discovery, and research applications that include, but are notlimited to, diagnosis or monitoring of a disease or condition (where theimage identifies a marker for the disease or condition), discovery ofdrug targets (where the a marker in the image may be targeted for drugtherapy), drug screening (where the effects of a drug are monitored by amarker shown in the image), determining drug susceptibility (where drugsusceptibility is associated with a marker) and basic research (where isit desirable to measure the differences between cells in a sample).

In certain embodiments, two different samples may be compared using theabove methods. The different samples may be composed of an“experimental” sample, i.e., a sample of interest, and a “control”sample to which the experimental sample may be compared. In manyembodiments, the different samples are pairs of cell types or fractionsthereof, one cell type being a cell type of interest, e.g., an abnormalcell, and the other a control, e.g., normal, cell. If two fractions ofcells are compared, the fractions are usually the same fraction fromeach of the two cells. In certain embodiments, however, two fractions ofthe same cell may be compared. Exemplary cell type pairs include, forexample, cells isolated from a tissue biopsy (e.g., from a tissue havinga disease such as colon, breast, prostate, lung, skin cancer, orinfected with a pathogen etc.) and normal cells from the same tissue,usually from the same patient; cells grown in tissue culture that areimmortal (e.g., cells with a proliferative mutation or an immortalizingtransgene), infected with a pathogen, or treated (e.g., withenvironmental or chemical agents such as peptides, hormones, alteredtemperature, growth condition, physical stress, cellular transformation,etc.), and a normal cell (e.g., a cell that is otherwise identical tothe experimental cell except that it is not immortal, infected, ortreated, etc.); a cell isolated from a mammal with a cancer, a disease,a geriatric mammal, or a mammal exposed to a condition, and a cell froma mammal of the same species, preferably from the same family, that ishealthy or young; and differentiated cells and non-differentiated cellsfrom the same mammal (e.g., one cell being the progenitor of the otherin a mammal, for example). In one embodiment, cells of different types,e.g., neuronal and non-neuronal cells, or cells of different status(e.g., before and after a stimulus on the cells) may be employed. Inanother embodiment of the invention, the experimental material is cellssusceptible to infection by a pathogen such as a virus, e.g., humanimmunodeficiency virus (HIV), etc., and the control material is cellsresistant to infection by the pathogen. In another embodiment, thesample pair is represented by undifferentiated cells, e.g., stem cells,and differentiated cells.

The images produced by the method may be viewed side-by-side or, in someembodiments, the images may be superimposed or combined. In some cases,the images may be in color, where the colors used in the images maycorrespond to the labels used.

Cells any organism, e.g., from bacteria, yeast, plants and animals, suchas fish, birds, reptiles, amphibians and mammals may be used in thesubject methods. In certain embodiments, mammalian cells, i.e., cellsfrom mice, rabbits, primates, or humans, or cultured derivativesthereof, may be used.

Computer Systems

The invention also provides a computer system that is configured toimplement the methods of the disclosure. The system can include acomputer server (“server”) that is programmed to implement the methodsdescribed herein. FIG. 21 depicts a system 1600 adapted to enable a userto detect, analyze, and process images of samples. The system 1600includes a central computer server 1601 that is programmed to implementexemplary methods described herein. The server 1601 includes a centralprocessing unit (CPU, also “processor”) 1605 which can be a single coreprocessor, a multi core processor, or plurality of processors forparallel processing. The server 1601 also includes memory 1610 (e.g.random access memory, read-only memory, flash memory); electronicstorage unit 1615 (e.g. hard disk); communications interface 1620 (e.g.network adaptor) for communicating with one or more other systems; andperipheral devices 1625 which may include cache, other memory, datastorage, and/or electronic display adaptors. The memory 1610, storageunit 1615, interface 1620, and peripheral devices 1625 are incommunication with the processor 1605 through a communications bus(solid lines), such as a motherboard. The storage unit 1615 can be adata storage unit for storing data. The server 1601 is operativelycoupled to a computer network (“network”) 1630 with the aid of thecommunications interface 1620. The network 1630 can be the Internet, anintranet and/or an extranet, an intranet and/or extranet that is incommunication with the Internet, a telecommunication or data network.The network 1630 in some cases, with the aid of the server 1601, canimplement a peer-to-peer network, which may enable devices coupled tothe server 1601 to behave as a client or a server. A microscope can be aperipheral device 1625 or a remote computer system 1640.

The storage unit 1615 can store files, such as individual images, timelapse images, data about individual cells, data about individualbiomarkers, images showing a pattern of binding of capture agents to asample, or any aspect of data associated with the invention. The datastorage unit 1615 may be coupled with data relating to locations ofcells in a virtual grid.

The server can communicate with one or more remote computer systemsthrough the network 1630. The one or more remote computer systems maybe, for example, personal computers, laptops, tablets, telephones, Smartphones, or personal digital assistants.

In some situations the system 1600 includes a single server 1601. Inother situations, the system includes multiple servers in communicationwith one another through an intranet, extranet and/or the Internet.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) computer readable medium (or software) stored on anelectronic storage location of the server 1601, such as, for example, onthe memory 1610, or electronic storage unit 1615. During use, the codecan be executed by the processor 1605. In some cases, the code can beretrieved from the storage unit 1615 and stored on the memory 1610 forready access by the processor 1605. In some situations, the electronicstorage unit 1615 can be precluded, and machine-executable instructionsare stored on memory 1610. Alternatively, the code can be executed on asecond computer system 1640.

Aspects of the systems and methods provided herein, such as the server1601, can be embodied in programming Various aspects of the technologymay be thought of as “products” or “articles of manufacture” typicallyin the form of machine (or processor) executable code and/or associateddata that is carried on or embodied in a type of machine readable medium(e.g., computer readable medium). Machine-executable code can be storedon an electronic storage unit, such memory (e.g., read-only memory,random-access memory, flash memory) or a hard disk. “Storage” type mediacan include any or all of the tangible memory of the computers,processors or the like, or associated modules thereof, such as varioussemiconductor memories, tape drives, disk drives and the like, which mayprovide non-transitory storage at any time for the software programming.All or portions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical, and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless likes, opticallinks, or the like, also may be considered as media bearing thesoftware. As used herein, unless restricted to non-transitory, tangible“storage” media, terms such as computer or machine “readable medium”refer to any medium that participates in providing instructions to aprocessor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, tangible storage medium,a carrier wave medium, or physical transmission medium. Non-volatilestorage media can include, for example, optical or magnetic disks, suchas any of the storage devices in any computer(s) or the like, such maybe used to implement the system. Tangible transmission media caninclude: coaxial cables, copper wires, and fiber optics (including thewires that comprise a bus within a computer system). Carrier-wavetransmission media may take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include, for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame,any other physical storage medium with patterns of holes, a RAM, a ROM,a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables, or linkstransporting such carrier wave, or any other medium from which acomputer may read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

The results of the sample staining or labeling can be presented to auser with the aid of a user interface, such as a graphical userinterface.

Kits

In some aspects, the disclosure herein provides for kits. The kits cancomprise any number of compositions to perform the methods of thedisclosure, each of which have been described herein. For example, a kitmay comprise at least one capture agent. The capture agent can be anantibody, an aptamer, or an oligonucleotide probe. The capture agent canbe custom-made to specifically bind to a desired target. For example, auser may custom-order one or more capture agents to be included in thekit. In some cases, the capture agents can be sold separately. Thecapture agents can specifically bind to a target molecule of interest.Additionally or alternatively, capture agents can be ordered as a panel(i.e., a pre-determined selection of capture agents). The panel can bespecific for a particular type of disease (e.g., cancer) or a particularsub-type of a disease (e.g., colon cancer). A kit of the disclosure canalso include one or more oligonucleotides. The oligonucleotides cancomprise a first strand and a double strand, as described herein. Theoligonucleotides can be provided as single-stranded oligonucleotides oras double-stranded oligonucleotides. In the latter case, the kit caninclude reagents and/or instructions for annealing the first strand andthe second strand of oligonucleotides to produce double-strandedoligonucleotides. The single- or double-stranded oligonucleotides can beconjugated to the capture agents or can be provided unconjugated. In thelatter case, reagents can be included in the kit for conjugating thedouble-stranded oligonucleotides to the capture agents (e.g., reagentsto perform Click chemistry). In some cases, the kit may provide aplurality of oligonucleotides wherein each of the first strands is thesame and each of the second strands is different. The kit can furthercomprise any nucleotide mixture disclosed herein. Nucleotide mixturescan comprise any combination of fluorescent nucleotides, unlabelednucleotides, reversible terminator nucleotides, and the like. Generally,the nucleotide mixture provided in the kit will be compatible with theprovided oligonucleotides. The kit can further comprise, withoutlimitation, a polymerase for performing primer extension, a reagent forinactivating a signal (e.g., TCEP), a blocking solution (e.g.,iodoacetamide solution), and any buffer or solution suitable to performthe methods herein. The kit can comprise any reagent for preparing asample for labeling such as a fixative (e.g., formaldehyde) or reagentsfor embedding a sample (i.e., paraffin wax). The kit can furthercomprise a control sample for comparison with a test sample. The controlsample can be a healthy sample or a diseased sample. The control samplemay be matched to the tissue or cell type under investigation or to thedisease being studied. In some cases, the control sample may be apositive control or a negative control.

EMBODIMENTS

A method for analyzing a planar sample is provided. In certainembodiments, the method comprises: (a) incubating the planar sample witha capture agent under conditions by which the capture agent specificallybinds to complementary sites in the planar sample, wherein: (i) thecapture agent is linked to a double-stranded oligonucleotide thatcomprises a first strand and a second strand; (ii) the capture agent islinked to a double-stranded oligonucleotide by the 5′ end of the firststrand; and (iii) the 3′ end of the first strand is recessed relative tothe 5′ end of the second strand, thereby producing an overhang; (b)crosslinking the capture agent to planar sample; (c) contacting theplanar sample with a polymerase and a nucleotide mix, thereby adding oneor more nucleotides to the overhang; and/or contacting the planar samplewith a mixture of short oligonucleotides, some of which may be labelledor not, and a DNA ligase and (d) reading a fluorescent signal generatedby addition of the one or more nucleotides to the overhang usingfluorescence microscopy, thereby producing an image showing the patternof binding of the capture agent to the planar sample. In someembodiments, after the sample has been read, this method may involveremoving the fluorescent moiety and deprotecting an added fluorescentnucleotide, thereby allowing the method to be repeated.

In any embodiment, step (c) may comprise contacting the planar samplewith a polymerase and a nucleotide mix that comprises a fluorescentnucleotide, thereby adding the fluorescent nucleotide to the overhang,or contacting to planar sample with one or more fluorescently labeledoligonucleotides, thereby adding a fluorescently labeled oligonucleotideto the overhang; and step (d) comprises reading a fluorescent signalgenerated by addition of the fluorescent nucleotide or oligonucleotideto the overhang. In this embodiment, the fluorescent signal may be:emitted directly from the added nucleotide or oligonucleotide, a FRETsignal generated by energy transfer between two fluorescent nucleotidesthat are added to the overhang or a FRET signal generated by energytransfer between a first fluorescent nucleotide added to overhang and asecond fluorescent nucleotide that is present in the second strand.

In some embodiments, extension of the first strand removes a quencherfrom a quenched fluorescently labeled oligonucleotide that is hybridizedto the second strand, downstream from the first strand.

In any embodiment, the sample may be a formalin-fixed, paraffin-embedded(FFPE) section.

Also provided herein is a capture agent that is linked to adouble-stranded oligonucleotide, wherein: (i) the double-strandedoligonucleotide comprises a first strand and a second strand; (ii) thecapture agent is linked to the 5′ end of the first strand; and (iii) the3′ end of the first strand is recessed relative to the 5′ end of thesecond strand, thereby producing an overhang or (iiii) the 5′ end isrecessed relative to the 3′ end of the second strand, there producingand overhang

Also provided herein is a capture agent composition comprising aplurality of capture agents that recognize different complementarysites, wherein: each of the capture agents is linked to adouble-stranded oligonucleotide that comprises a first strand and asecond strand; the capture agents are linked to a double-strandedoligonucleotide by the 5′ end of first strand; the 3′ end of the firststrand in each of the double-stranded oligonucleotides is recessedrelative to the 5′ end of the second strand, thereby producing anoverhang; and the overhang is different for each of the capture agents.Alternatively, the 5′ end is recessed relative to the 3′ end of thesecond strand, there producing and overhang that is specific to eachcapture agent. In this embodiment, the sequence of the first strand maybe the same for each of the capture agents; and the sequence of thesecond strand may be different for each of the capture agents.

In these embodiments, the overhangs may be of the formula3′-N_(4n)N₁/N₂/N₃, wherein N₁, N₂, N₃ and N₄ are different nucleotidesselected from G, A, T and C and n is 1 or more, or the formula3′-YN₁/N₂-5′, optionally followed by short stretch of random nucleotideson the 5′ end to increase the overall polymerase residence on the DNAduplex, wherein Y is composed of alternating stretches of bases N₃ andN₄, and wherein N₁, N₂, N₃ and N₄ are different nucleotides selectedfrom G, A, T and C.

A method for analyzing a tissue sample is also provided. In theseembodiments, the method may comprise (a) incubating a planar sample witha capture agent composition of a prior embodiment under conditions bywhich the capture agents specifically bind to sites in the planarsample; (b) crosslinking capture agents to planar sample; (c) contactingthe planar sample with a polymerase and either an incomplete nucleotidemix or a nucleotide mix that comprises a reversible terminatornucleotide and/or a ligase and a labeled oligonucleotide; and (d)reading, using fluorescence microscopy, a fluorescent signal generatedby addition a nucleotide to some but not all of the capture agents.

In this embodiment, the method may comprise: (c) contacting the planarsample with a polymerase and: (i) a nucleotide mix that comprisesfluorescent nucleotides that are complementary to N₁, N₂ and N₃ and areversible terminator nucleotide that is complementary to N₄ or (ii) anucleotide mix that comprises fluorescent nucleotides that arecomplementary to N₁, and N₂, an unlabeled nucleotide that iscomplementary to N₃, and no nucleotide that is complementary to N₄,thereby adding fluorescent nucleotides onto the double-strandedoligonucleotides of some but not all of the capture agents. This stepcan also be done by ligation, i.e., by contacting the planar sample witha labeled oligonucleotide (or a mixture of the same), where addition ofthe labeled oligonucleotide depends on the underlying sequence of theoverhang. This method also comprises: (d) reading, using fluorescencemicroscopy, a fluorescent signal generated by addition of a fluorescentnucleotide to some but not all of the capture agents. In theseembodiments, the overhangs may be of the formula 3′-N_(4n)/N₂/N₃,wherein N₁, N₂, N₃ and N₄ are different nucleotides selected from G, A,T and C and n is 1 or more; and step (c) comprises contacting the planarsample with a polymerase and a nucleotide mix that comprises fluorescentnucleotides that are complementary to N₁, N₂ and N₃ and a reversibleterminator nucleotide that is complementary to N₄. This step can also beimplemented by addition of a labeled oligonucleotide using a ligase. Inthese embodiments, the method may comprise (e) inactivating thefluorescent signal, deprotecting the reversible terminator nucleotideand blocking the sample; and (f) repeating steps (c) and (d). In somecases, step (f) may comprise repeating steps (c), (d) and (e) multipletimes. Alternatively, the overhangs may be of the formula 3′-YN₁/N₂-5′,optionally followed by short stretch of random nucleotides on the 5′ endto increase the overall polymerase residence on the DNA duplex, whereinY is composed of alternating stretches of bases N₃ and N₄, and whereinN₁, N₂, N₃ and N₄ are different nucleotides selected from G, A, T and C.In these embodiments, the method may further comprise (e) inactivatingthe fluorescent signal and contacting the planar sample with apolymerase and an unlabeled nucleotide that is complementary to N₄; and(f) repeating steps (c) and (d). In some cases, step (f) may compriserepeating steps (c), (d) and (e) multiple times.

In some embodiments, the double-stranded oligonucleotides each comprisea fluorescently labeled oligonucleotide hybridized to the second stranddownstream from first strand, wherein the fluorescently labeledoligonucleotide comprises a quencher and extension of the first strandremoves the quencher from some but not all of the quenched fluorescentlylabeled oligonucleotides, thereby generating a fluorescent signal forsome but not all of the capture agents.

Example I Materials and Methods

Spleen cells fixed in 2% formaldehyde, permeablized and stored inmethanol at −80 were spun from methanol, resuspended and washed withbuffer 4 (10 mM Tris & 0.5, 10 mM MgCl2, 150 mM NaCl, 0.1% Triton x100)for 5 min on a rotator. To block against non-specific binding ofab-oligonucleotide complexes cells were further spun, resuspended in 1ml PBS, 0.5% BSA (SM) and supplemented up to additional 0.5M NaCl (0.9ml SM+100 ul 5M NaCl). 20 ul of sheared ssDNA (10 mg/ml), 50 ul of mouseIgG (10 mg/ml) and 20 ul of 0.5M EDTA were further added to 1 ml ofcells and the mix was incubated for 30 min on a rotator. For stainingcells were redistributed into 30 250 ul tubes (PCR strip tubes is aconvenient choice for that matter) with premade antibody/oligonucleotidecomplexes (0.2 ug of CD45-146 complex was annealed with 1 ul of specificoligonucleotide (147 etc) per tube 30 min at 40 C) and incubated for 1 hwith rotation. Cells were washed in (PBS, 0.1% Triton 0.5M salt 5 mMEDTA) twice, placed on poly-lysine treated glass coverslips, allowed tostand/attach for 10 min and further fixed with 5 mM BS3 (7.4 mg per 4ml) in PBS, 0.1% Triton, 0.5M NaCl, 5 mM EDTA for 1 hour.

Staining was rendered in cycles. For odd cycles (1, 3, 5, 7, 9, 11, 13,15) coverslips were incubated for 2 min in dG/dU mix (150 nM dG, 150 nMdUssCy5, 150 nM dCssCy3, 25 ul NEB exo− Klenow per ml in buffer #4 (10mM Tris 7.5, 0.5M NaCl, 0.1% Triton x100, 10 mM MgCl2)), washed twicewith 405 (buffet #4 supplemented up to 0.65M NaCl); and imaged byconfocal microscopy. Following imaging the fluorophores were cleaved offcells by incubation in 50 mM TCEP for 2 min in buffer 405E (10 mM Tris7.5, 0.5M NaCl, 0.1% Triton x100, 5 mM EDTA). After cleavage cells werewashed in 405E and blocked for for 1 min in iodoacetamide solution(FRESHLY made 100 mM iodoacetamide in buffer 405E). The blockingsolution was removed by two washes with buffer #4. Before proceeding tonext cycle cells were again imaged by confocal microscopy. Even cycles(2, 4, 6, 8, 10, 12, 14) were performed same as odd cycles except forsubstitution of dG with dA in labeling step and extension of cleavage to4 min at room temperature.

Preliminary Data.

To explore the possibility of in situ staining by primer extensionexpression of CD4 was visualized in mouse spleen cells in suspension(FIG. 11) or immobilized on a slide. (FIG. 12). To visualize the Tlymphocytes spleen cells were co-stained with conventional TcrB-Ax488antibody. Both samples were stained with CD4 antibody conjugated tooligonucleotide duplex as in (FIG. 11 A). No Klenow polymerase was addedin control samples which results in no separation of TcrB positiveT-cells into subsets (FIG. 11 B). When Klenow polymerase was supplied.CD4 positive T-cells could be observed as a Cy5 positive subset of TcrBpositive T-cells (FIG. 11 C and FIG. 12). Clear membrane stainingpattern was observed by confocal imaging of cells stained on-slide (FIG.12 A). Taken together this data shows that on-slide primer extensionreaction can be used for rendering the capture agent binding pattern

FIG. 11. Flow cytometric analysis of mouse spleen cells stained byprimer extension. Mouse spleen cells were fixed and permeabilized withmethanol as done for intracellular protein staining. Cells wereco-stained with conventional TcrB-Ax488 antibody and CD4 antibodyconjugated to oligonucleotide duplex as in (A). After staining cellswere either incubated in extension buffer with dUTP-Cy5 without (B) orwith (C) Klenow exo⁻ polymerase. Note that TcrB positive T-cells in (B)are indicated by Ax-488 staining. Dependent upon the addition of Klenow,TcrB positive CD4 positive T-cells are seen as a Cy5 positive subset ofTcrB positive T-cells in (C).

FIG. 12. On-slide analysis of mouse spleen cells stained by primerextension. Mouse spleen cells were fixed and permeabilized with methanolas done for intracellular protein staining. Cells were attached topoly-Lysine coated slide and co-stained with conventional TcrB-Ax488antibody and CD4 antibody conjugated to oligonucleotide duplex as inFIG. 12 A. After staining, cells were incubated in extension buffer withdUTP-Cy5 Klenow exo⁻ polymerase and visualized by confocal microscopy.Shown are DIC image in C, Cy5 channel in A, Ax488 channel in B andmerged Ax488 and Cy5 channels in D. Note that only a subset ofTcrB-Ax488 positive T-cells in (B) are rendered Cy5 positive CD4positive T-cells by primer extension as seen in (A). The membranepattern of CD4 points to specificity of staining by primer extension asit takes place at a particular expected subcellular localization.

To prove the possibility of multiplexed detection of several antigens byprimer extension, the expression of CD4 and CD8 was co-analyzed in mousespleen cells immobilized on a slide by Method 1 and, specifically, themultiplexing approach based on “reversible terminators”. The cells weresimultaneously stained by CD4 and CD8 antibodies conjugated tooligonucleotide duplexes as in (FIG. 14, A) simultaneously. Two cyclesof rendering were performed such that CD8 was visualized in the firstcycle (FIG. 14, C) and CD4 in the second (FIG. 14, D). Cells werecounterstained with TcrB-Ax488 to delineate T-lynphocytes in the spleencells. As expected CD4 positive cells were rendered as a subset of TcrBpositive T-cells mutually exclusive with CD8-positive subset ofT-lymphocytes (FIG. 14, A-D). The data suggests that rendering antibodystaining by polymer (DNA-duplex) extension is an approach enablingsensitive antigen detection and multiplexing.

FIG. 13. Two cycle analysis of CD4 and CD8 staining in mouse spleenusing Method 1 with reversible terminators. Mouse spleen cells werefixed and permeabilized with methanol as done for intracellular proteinstaining. Cells were attached to poly-Lysine coated slide and co-stainedwith conventional TcrB-Ax488 antibody and a mixture of CD4 and CD8antibodies conjugated to oligos as indicated on (A). For the first cycleof staining the cells were incubated in extension buffer with Illuminareversible terminators and Klenow exo⁻ polymerase and visualized byconfocal microscopy (C). Following the imaging after the first cycle,cells were destained by Illumina cleavage buffer containing TCEP.Following destaining-terminator reactivation, cells were again incubatedin extension buffer with Illumina reversible terminators and Klenow exo⁻polymerase and visualized by confocal microscopy (D) Note that fourT-cells identified by high levels of TcrB and marked by four whitearrows on (B). It becomes evident after the first cycle of staining thattwo of these cells are CD8a positive (marked by purple arrows on (C).Second cycle of staining reveals that the other two cells are CD4positive (marked by green arrows on (D). The expected mutual exclusivityof CD4 and CD8a as well as membrane pattern of incorporated labelednucleotide further supports the specificity of staining by cycles ofprimer extension.

The “missing base” multiplexing approach was tested on a model ofheterogeneous tissue containing multiplicity of distinct cellularsubsets (FIG. 14). To this end leukocytes from homogenized mouse spleenwere divided into 30 samples. 30 different versions of CD45 were made byconjugating purified CD45 to common upper strand oligonucleotide andthen separately annealing 30 different lower strand oligonucleotidesdesigned to create overhangs that can be sequentially rendered (two percycle) in the multiplexed version of “missing base approach”. Thesamples were individually stained (barcoded) by 30 CD45 antibodyconjugates, the unbound CD45 was washed off the barcoded samples weremixed and attached to a slide. The staining of this mixture ofpseudotyped cells was rendered by “missing base” approach. Six firstcycles (12 populations, 2 red and green per cycle) as well asinactivation of fluorescence by cleaving the fluorophore off themodified base by TCEP between the cycles is shown on FIG. 15. As can beseen no same two cells are stained in each cycle and between the cyclesproving that on-cell primer extension reliably renders the specificantibody staining.

In order to test the performance and multiplexing capacity of “missingbase” method the following model approach was employed, as shown inFIGS. 14 and 15. Mouse CD45 antibody was chemically conjugated to an“upper strand” oligonucleotide (oligonucleotide id-146). The conjugatedantibody was further divided and separately annealed (by 30 minco-incubation at 40 C) to 30 different “lower strand”oligonucleotides—thus effectively creating 30 different species of CD45antibody. The 30 “lower strand” oligonucleotides were designed inaccordance with “missing base” strategy and in addition in such a waythat 2 antibodies could be rendered per cycle using two bases (dUTP anddCTP) reversibly (through s-s linker) coupled with distinct fluorophores(Cy5 and Cy3). 30 samples of homogenized mouse spleen have been“barcoded” with these CD45-oligonucleotide duplex complexes such waythat majority of cells in each sample became labeled with a particularCD45-upper/lower oligonucleotide combination. Following staining andwashing the samples were combined to mimic a tissue with 30 differentcellular subsets. The mixture was smeared on a slide and rendered bycycling staining with a “missing base” approach such that two subsetsper staining cycle were co-visualized on different imaging channels.

Example 2

Antibody Signal Amplification In Situ with Rolling Circle Amplification

Materials and Methods

Rat anti-mouse B220 antibody conjugate to oligonucleotide 146v2 wasprepared as described. The conjugate were hybridized to a padlockoligonucleotide (PatgctaccgttAATTATTACTGAAACATACACTAAAGATAACATTAttctgcaag: SEQ NO:125) that is designed to form a circular hybrid on146v2. Mouse spleen cells were stained with either of the conjugates andthen those cells that were stained with the padlock construct wereincubated with T4 RNA ligase (NEB) in manufacturer's ligation buffer at37 C for 1 hour and then with phi29 polymerase and dNTP mix for 15minutes. Cells were washed 3 times with PBS and then incubated with 10nM RCA product detection oligonucleotide (TGAAACATACACTAAAGA; SEQ IDNO:126) for 10 minutes. After that, cells were incubated withfluorescent dUTP-Cy5 (Jena Biosciences) was incorporated into the cellsby incubating with 200 nM dUTP-Cy in buffer #4 and 1 ul of exo− Klenowpolymerase (Thermo Scientific). An aliquot of cells was left out and notsubject to the rolling circle amplification (RCA) step and then used asa reference to assess the effect of RCA on staining.

Results

The efficiency of rolling circle amplification of multiplexing DNAbarcode attached to the antibody for enhancing antibody staining wastested. A special antibody-DNA conjugate based on anti-B220 antibodythat contained a circularized ‘padlock’ oligonucleotide annealed to thelinker (146v2) oligonucleotide hybrid was assembled and mouse cells werestained with it. After staining, the padlock oligonucleotide was ligatedwith T4 ligase and amplified using the rolling circle protocol withphi29 polymerase, resulting in a long repetitive DNA stretch attached toeach antibody that contained the repetitive sequence complementary tothe detection primer. After annealing of the primer, multiple moleculesof dUTP-Cy5 could be incorporated into the amplified DNA molecule, dueto its repetitive nature. FIG. 16 panels A-E schematically illustratethis method. FIG. 16 panels F-G shows that the cells staining with therolling circle amplification is much stronger than without it.

Example 3

Co-Detecting 22 Antigens on Dispersed Spleen Cells

Materials and Methods

Antibody conjugates were prepared using the following protocol.Antibodies were subject to partial reduction of disulfide by 30 minincubation at room temperature with TCEP (final concentration 1 mM) inPBS pH 7.4. The antibodies were purified from TCEP by buffer exchange onBioGel P-30 spin-columns saturated with conjugation buffer (PBS pH 7.0).Oligonucleotide 146v2 (5′ Maleimide-ATAGCAGTCCAGCCGAACGGTAGCATCTTGCAGAA; SEQ ID NO: 27) bearing a protected maleimide group wereordered from Trilink Inc. To prepare for covalent crosslinking toantibodies per instruction from manufacturer the maleimide groupresiding on an oligonucleotide was de-protected/activated by Adlerreaction (4 h at 90 C in toluene). Toluene was removed from theoligonucleotide by several washes in absolute ethanol. Activatedoligonucleotides were dissolved in conjugation buffer and mixed withreduced antibodies at a molar ratio of 50:1. Sodium Chloride was addedto conjugation reaction to final concentration of 1M. Conjugationreaction was allowed to proceed for 1 h. To remove the unboundoligonucleotide the conjugated antibodies were filtered 4 times onmolecular weight cutoff filters (Amicon 50 KDa). Final wash and storagewere performed in phosphate buffer with 0.5M sodium chloride and 0.1%Tween-20.

To assemble DNA duplex tag 0.2 ug of conjugated antibodies was mixedwith 100 pmoles of bottom strand oligonucleotide in phosphate bufferwith 0.6M Sodium Chloride and incubated for 30 min at 40° C.

v2_C_cycle1 SEQ ID NO: 128: TTTTGTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle2SEQ ID NO: 129: TTTTGtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle3SEQ ID NO: 130: TTTTGCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle4SEQ ID NO: 131: TTTTGTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle5SEQ ID NO: 132: TTTTGCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle6SEQ ID NO: 133: TTTTGtttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle7SEQ ID NO: 134:  TTTTGcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle8(SEQ ID NO: 135) TTTTGttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle9(SEQ ID NO: 136) TTTTGcttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_C_cycle10(SEQ ID NO: 137) TTTTGtcttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGzv2_C_cycle11 (SEQ ID NO: 138)TTTTGcctcttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle1SEQ ID NO: 139: TTTTATTCTGCAAGATGCTACCGTTCGGz v2_U_cycle2SEQ ID NO: 140: TTTTAtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle3SEQ ID NO: 141: TTTTACtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle4SEQ ID NO: 142: TTTTATCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle5SEQ ID NO: 143: TTTTACcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle6SEQ ID NO: 144: TTTTAtttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle7SEQ ID NO: 145: TTTTAcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle8(SEQ ID NO: 146) TTTTAttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle9(SEQ ID NO: 147) TTTTActtcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz v2_U_cycle10(SEQ ID NO: 148) TTTTAtcttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGzv2_U_cycle11 (SEQ ID NO: 149)TTTTAcctcttcctttCcTCtTTCTGCAAGATGCTACCGTTCGGz

Mouse spleen and bone marrow cells were prepared according to standardprocedure and fixed in 2% formaldehyde for 10 min at room temperature.Following fixation, cells were spun and either stored frozen at −80 inPBS with 5% DMSO or permeabilized by incubation in ice-cold methanol for10 min. and further stored at −80° C. in methanol.

Before staining stored cells were washed with SM (0.5% BSA in PBS,5mMEDTA) once and blocked for 30 min at room temperature in stainingbuffer (0.6M NaCl, 0.5% BSA, 50 ug/ml rat IgG, 200 ug/ml ssDNA, 5mMEDTA,3 nmoles per ml of blocking oligonucleotideTTTTccctctcctcttcctttCcTCt-ddC in phosphate buffer pH 7.4). In casefrozen sections were used tissue sections were picked by warm coverslipsand immediately placed into dry ice without allowing the section to dry.Coverslips with sections were dipped for 30 sec into ethanol pre-chilledto dry-ice temperature, and transferred to SME with 4% formaldehyde for20 min After that the fixed sections were washed twice in SM and furtherblocked in staining buffer for 30 min. Mouse spleen cells were stainedin staining buffer for 2-3 h at room temperature with a mixture ofconjugated antibodies taken at 0.2 ug of each antibody per 100 ul ofsolution. After staining cells were washed twice with SM05 (SMsupplemented with NaCl up to 0.65M final concentration), then wereallowed to adhere to poly-L-lysin coated coverslips and further fixed tocoverslip surface by 20 min incubation with 5 mM BS3 crosslinker in PBS.Following methanol fixation/permeabilization cells were washed once withSM05, allowed to adhere to coverslip surface and further fixed tocoverslip surface by 20 min incubation with 5 mM BS3 crosslinker in PBS.If frozen sections were used—following staining sections were washedtwice by SM05 and fixed by 20 min incubation with 5 mM BS3 crosslinkerin PBS. Following staining procedure and converting into planar form (incase of suspension cells) all kinds of samples were subjected to similarABseq rendering protocol.

Coverslips with cells were washed twice with buffer 4 (10 mM Tris pH6.5, 10 mM MgCl2, 150 mM NaCl, 0.1% Triton x100).

Staining was rendered by iterative incubation with polymerase reactionmixes. In odd cycles (1, 3, 5 . . . )-cells were incubated for 2 min inG-mix (150 nM dG, 150 nM dUssCy5, 150 nM dCssCy3, 25 ul NEB exo− Klenowper ml in buffer 4); wash 3 times with 405 (buffet 4 without MgCl andsupplemented with NaCl up to final 0.65M); photographed; incubated 2 minin 50 mM TCEP in buffer 405; washed twice with 405; photographed;incubated for 1 min in freshly made 100 mM iodoacetamide in buffer 405;washed three times with buffer 4. In even cycles (2, 4, 6 . . . )-cellswere incubated for 2 min in A-mix (150 nM dATP, 150 nM dUTPssCy5, 150 nMdCTPssCy3, 25 ul NEB exo− Klenow per ml in buffer 4); wash 3 times with405 (buffet 4 without MgCl and supplemented with NaCl up to final0.65M); photographed; incubated 4 min in 50 mM TCEP in buffer 405;washed twice with 405; photographed; incubated for 1 min in freshly made100 mM iodoacetamide in buffer 405; washed three times with buffer #4.Reversibly labelled fluorescent nucleotide triphosphates were customsynthesized by Jena Bioscience.

Results

ABseq was used to explore the variety of cellular subsets in mousespleen and bone marrow using 22-antibody panel. Isolated spleen and bonemarrow cells were barcoded by whole cell staining with NHS-PacBlu andNHS-Ax-488 dyes, mixed, stained with a panel of 22 antibodies taggedwith DNA duplexes, attached to slide and rendered by ABseq in 11 primerextension and imaging iterations (FIG. 17). Conspicuously 22-colormarker expression data on pseudocolored image bearing all markerexpressing data proved to be impossible to parse visually due toproximity of colors in multi-color palette (FIG. 17, bottom rightpanel).

Example 4 Multipanel Design with Spacers Materials and Methods

Antibody conjugation, cell staining and rendering was performedfollowing the same experimental procedures as in section 4 (Co-detecting22 antigens on dispersed spleen cells). Nine aliquots of spleen cellswere stained separately with a different CD45 antibody-DNA conjugate.Conjugates for each panel were formed in the following way.

Panel1: CD45 conjugated to 146v2 (5′Maleimide-ATAGCAGTCCAGCCGAACGGTAGCATCTTGCAGAA (SEQ ID NO:  174) and forming a DNA duplex with: 1. (SEQ ID NO: 150)TTTTATTCTGCAAGATGCTACCGTTCGG-dideoxyC 2. (SEQ ID NO: 151)TTTTAtTTCTGCAAGATGCTACCGTTCGGz-dideoxyC 3. (SEQ ID NO: 152)TTTTACtTTCTGCAAGATGCTACCGTTCGGz-dideoxyCPanel2 CD45 conjugated to 146v2-ddC (5′Maleimide-ATAGCAGTCCAGCCGAACGGTAGCATCTTGCAGAA-dideoxyC)(SEQ ID NO: 153)  and forming a DNA duplex with: 4. (SEQ ID NO: 154)TTTTAGCGATTAAGCGTGAACTTCTGCAAGATGCTACCGTTCGG- dideoxyC 5.(SEQ ID NO: 155) TTTTAtGCGATTAAGCGTGAACTTCTGCAAGATGCTACCGTTCGGz-dideoxyC 6. (SEQ ID NO: 156)TTTTACtGCGATTAAGCGTGAACTTCTGCAAGATGCTACCGTTCGGz- dideoxyCPanel3 CD45 conjugated to 146v2-ddC (5′Maleimide-ATAGCAGTCCAGCCGAACGGTAGCATCTTGCAGAA-dideoxyC)(SEQ ID NO: 157) and forming a DNA duplex with: 7. (SEQ ID NO: 158)TTTTACGCTAATTCGCACTTGTTCTGCAAGATGCTACCGTTCGG- dideoxyC 8.(SEQ ID NO: 159) TTTTAtCGCTAATTCGCACTTGTTCTGCAAGATGCTACCGTTCGGz-dideoxyC 9. (SEQ ID NO: 160)TTTTACtCGCTAATTCGCACTTGTTCTGCAAGATGCTACCGTTCGGz- dideoxyC

After staining the cells were washed with washed twice with buffer 4 (10mM Tris pH 6.5, 10 mM MgCl2, 150 mM NaCl, 0.1% Triton x100) to removeunbound antibody-DNA conjugates and then the aliquots of cells weremixed together and attached to a lysine-coated coverslip. Antigenstaining was rendered in the following sequence of incubations:dGTP+dUTP-Cy5->dATP+dUTP-Cy5->dGTP+dUTP-Cy5->Incubation with 1 uMspacer1 (GTTCACGCTTAATCGC; SEQ ID NO:161) in buffer #4 for 20minutes->dGTP+dUTP-Cy5->dATP+dUTP-Cy5->dGTP+dUTP-Cy5->Incubation with 1uM spacer2 (CGCTAATTCGCACTTG; SEQ ID NO:162) in buffer #4 for 20minutes->dGTP+dUTP-Cy5->dATP+dUTP-Cy5->dGTP+dUTP-Cy5 Imaging,fluorophore inactivation with 50 mM TCEP pH 7.0 and background blockingwith iodoacetamide were performed after each step of rendering.

Results

Due to polymerase misincorporation errors the signal intensity ofrendering by ABseq is expected to fall with increasing cycle numbers asobserved in other studies on development of deep sequencing protocolsutilizing sequential addition of individual nucleotides. To circumventthat and to avoid the use of extensively long DNA fragments linked toantibody the following amendment to the design was tested (FIG. 18,panel A). Large antibody panels can be split into subpanels such thatthe extension reaction on these subpanels is precluded by termination ofthe upper strand oligonucleotide with ddC, propyl or any other 3′terminating group. After finishing the extension of each subpanel, thenext subpanel is activated by in situ hybridization of a short“activation” spacer, which does not bear any terminating moiety on its3′ end and thus initiates the consecutive cycles of primer extensions.This design was tested experimentally on 3 sequential 3-cycle panels (9extension cycles in total) (FIG. 18, panel B). Image quantificationshowed no significant reduction of ABseq rendering efficiency associatedwith on-slide hybridization of panel activating spacer oligonucleotidewas observed and no signal carryover between the individual panels (FIG.18, panel C).

Example 5 Multiplexed Single Molecule RNA Detection Materials andMethods

NALM and Jurkat cell lines were grown to a density of 1 million/ml,fixed with 1.6% formaldehyde for 10 minutes and then transferred toice-cold methanol. An aliquot of 200K cells was washed with PBSTR (PBS,0.1% Tween-20 and 1:1000 Rnasin) and transferred to a hybridizationbuffer (1×SSC, 10% formamide, 10% vanadyl-ribonucleotide complex, 10%polyvinylsulfonic acid). DNA probe mixture was added to the finalconcentration of 100 nM and incubated at 40 C for 1 hour. Cells werewashed 2 times with PBSTR at room temperature for 5 minutes and 2 timeswith a high salt buffer (4×SSC in PBSTR) at 40 degrees for 20 minutes,once again washed with PBSTR and transferred to a ligation solution (0.1ul T4 DNA ligase (New England Biosciences), 5 ul 10× T4 ligase buffer(New England Biosciences), 45 ul H2O). Ligation proceeded for 1 h at 37C. Then cells were transferred to amplification solution (1 ul of phi29polymerase (Thermo Scientific), 5 ul of 10× polymerase buffer (ThermoScientific), 1 ul of 10 mM dNTP mix, 43 ul of H₂O) and incubated at 30 Cfor 1 h. Cells were washed with PBSTR and incubated with 1 mM “RCAdetection” oligonucleotide for 10 minutes at 37 C and transferred toSequencing buffer (10 mM Tris pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.1%Triton x100, 1:50 Klenow polymerase (Thermo Scientific), 200 mM dUTP-Cy5(Jena Biosciences)). Cells were washed twice with high salt wash buffer(10 mM Tris pH 7.5, 10 mM MgCl2, 650 mM NaCl, 0.1% Triton x100) andimaged using a florescent microscope.

HLA-DR padlock1 (SEQ ID NO: 163)PACATTAaaatcctagcacagggactcAATTATTACTGAAACATACACTA AAGATApaHLA-DR splint-primer1 (SEQ ID NO: 164)ctcatcagcacagctatgatgaTAATGTTATCTT HLA-DR padlock2 (SEQ ID NO: 165)PACATTAtagaactcggcctggatgatAATTATTACTGAAACATACACTA AAGATAHLA-DR splint-primer2 (SEQ ID NO: 166) ctgattggtcaggattcagaTAATGTTATCTTHLA-DR padlock3 (SEQ ID NO: 167)PACATTAtcaaagctggcaaatcgtccAATTATTACTGAAACATACACTA AAGATAHLA-DR splint-primer2 (SEQ ID NO: 168) tggccaatgcaccttgagccTAATGTTATCTTHLA-DR padlock4 (SEQ ID NO: 169)PACATTAtgatttccaggttggctttgAATTATTACTGAAACATACACTA AAGATAHLA-DR splint-primer2 (SEQ ID NO: 170) atagttggagcgctttgtcaTAATGTTATCTTHLA-DR padlock5 (SEQ ID NO: 171)PACATTAtttcgaagccacgtgacattAATTATTACTGAAACATACACTA AAGATAHLA-DR splint-primer2 (SEQ ID NO: 172) ctgtggtgacaggttttccaTAATGTTATCTTRCA detect (SEQ ID NO: 173) CATACACTAAAGATAACAT

Results

An on-slide primer extension protocol was applied to detect singlemolecules of human HLADRA mRNA in NALM pro-B-cell line. Jurkat T-celllymphoma line was used as a negative control to assess the background.In order to enable single molecule mRNA detection, a signalamplification system was designed based on proximity ligation androlling circle amplification (RCA). Five pairs of probes were designedin a way that the two oligos of each pair were complementary to directlyadjacent 20-nt stretches of HLADRA mRNA and that the 3′ region of theupstream oligonucleotide served as a splint for circularization of thedownstream padlock oligonucleotide (FIG. 19, A) and also as a primer forthe rolling circle amplification. After the complex assembly the cellswere washed and treated with T4 DNA ligase to circularize the padlockoligonucleotide and the incubated with phi29 polymerase and dNTP mix tocarry out the rolling circle amplification (FIG. 19, B). Amplificationproducts were incubated with “RCA detect” oligonucleotide (FIG. 19, C)and then fluorescent dUTP-Cy5 was incorporated by a single baseextension with Klenow to polymerase (FIG. 19, D). Cells were washed andimaged with a fluorescent microscope. Images of NALM cells that expressHLADR show abundant punctate staining in the cytoplasm that correspondsto the RCA products (FIG. 19, E) and the Jurkat cells that are negativefor HLADR show very few puncta (FIG. 19, F), demonstrating the highspecificity of the proximity ligation-based detection of HLADRA mRNA.

1.-37. (canceled)
 38. A method of analyzing a planar sample, the methodcomprising: obtaining a labeled composition comprising: (a) a biologicalsample; (b) a plurality of capture agents; (c) a labeledoligonucleotide; and (d) a ligase; wherein, (i) the plurality of captureagents are linked to double-stranded oligonucleotides comprising a firststrand and a second strand, wherein the ligase is configured to add thelabeled oligonucleotide to the first strand or the second strand, and(ii) the plurality of capture agents are chemically cross-linkeddirectly to the biological sample; and reading a signal generated by thelabeled composition.
 39. The method of claim 38, wherein the pluralityof capture agents are cross-linked to the biological sample via abifunctional cross-linker.
 40. The method of claim 38, wherein theplurality of capture agents are cross-linked to the biological samplevia an amine-to-amine crosslinker.
 41. The method of claim 38, whereinthe plurality of capture agents are cross-linked to the biologicalsample by formaldehyde or a disuccinimidyl crosslinker.
 42. The methodof claim 38, wherein the biological sample is planar.
 43. The method ofclaim 38, wherein the biological sample is a tissue section.
 44. Themethod of claim 38, wherein the biological sample is a tissue biopsy.45. The method of claim 38, wherein the biological sample is aformalin-fixed paraffin embedded tissue section.
 46. The method of claim38, wherein the plurality of capture agents comprises a first captureagent linked to a first double-stranded oligonucleotide and a secondcapture agent linked to a second double-stranded oligonucleotide. 47.The method of claim 38, wherein the plurality of capture agentscomprises at least 10 capture agents, each linked to a differentdouble-stranded oligonucleotide.
 48. The method of claim 38, wherein thedouble-stranded oligonucleotides linked to the plurality of captureagents are at least 10 nucleotides in length.
 49. The method of claim38, wherein the plurality of capture agents are antibodies.
 50. Themethod of claim 49, wherein the antibodies are monoclonal antibodies.51. The method of claim 38, wherein the plurality of capture agents areaptamers or oligonucleotide probes.
 52. The method of claim 38, whereinthe double-stranded oligonucleotides comprises an overhang.
 53. Themethod of claim 38, wherein the labeled oligonucleotide comprises afluorescent label.
 54. The method of claim 38, wherein at least one ofthe plurality of capture agents comprises a fluorescent label.
 55. Themethod of claim 38, wherein the signal is a fluorescent signal.
 56. Themethod of claim 38, wherein reading comprises fluorescence microscopy.57. The method of claim 38, wherein the method further comprisesproducing an image showing the pattern of binding of the plurality ofcapture agents chemically cross-linked directly to the biologicalsample.